Profile of Bioactive Components and Antioxidant Activity of Aronia melanocarpa Fruits at Various Stages of Their Growth, Using Chemometric Methods.

Aronia (chokeberry, Aronia melanocarpa) is a valuable fruit that combines the health benefits of green tea and red wine and is gaining popularity worldwide. Aronia has a unique chemical composition with strong antioxidant properties, including anthocyanins and chlorogenic acids (CGAs). However, it remains unclear which specific compounds or groups are primarily responsible for the antioxidant properties of chokeberry. Therefore, an analysis of the antioxidant properties of aronia berries based on geographical region and their variability during ripening (from green to frostbitten fruit) was conducted. The fruits were collected from three locations for two years. The aim of our work was to identify the compounds responsible for the antioxidant properties of aronia berry extracts by using chemometric methods. The analyses of anthocyanins and CGAs were performed using HPLC-DAD, and the antioxidant capacity was assessed by FRAP and DPPH methods. The PCA analysis also considered variations in temperature and precipitation. The chemometric analysis revealed a strong correlation between radical-scavenging properties and the content levels of chlorogenic acids. The results obtained in this study show that unripe green chokeberry fruits exhibit the highest antioxidant properties, which can be attributed to the high content of CGAs at this stage.

Anti-Atherosclerotic Properties of Aronia melanocarpa Extracts Influenced by Their Chemical Composition Associated with the Ripening Stage of the Berries.

The high content of bioactive compounds in Aronia melanocarpa fruit offers health benefits. In this study, the anti-atherosclerotic effect of Aronia extracts was assessed. The impact on the level of adhesion molecules and the inflammatory response of human umbilical vein endothelial cells (HUVECs) was shown in relation to the chemical composition and the stage of ripening of the fruits. Samples were collected between May (green, unripe) and October (red, overripe) on two farms in Poland, which differed in climate. The content of chlorogenic acids, anthocyanins, and carbohydrates in the extracts was determined using HPLC-DAD/RI. The surface expression of ICAM-1 and VCAM-1 in HUVECs was determined by flow cytometry. The mRNA levels of VCAM-1, ICAM-1, IL-6, and MCP-1 were assessed using the quantitative real-time PCR method. The farms' geographical location was associated with the quantity of active compounds in berries and their anti-atherosclerotic properties. Confirmed activity for green fruits was linked to their high chlorogenic acid content.

Solid state NMR study of the bioenhancer piperine, supported by GIPAW DFT calculations.

Piperine, an alkaloid found in black pepper fruits, has the properties of promoting the absorption of other substances (e.g. curcumin), therefore it is used in solid forms of dietary supplements as an additive increasing bioavailability. The aim of the study was to analyze piperine in a solid state and then to use it in the analysis of solid dietary supplements with the addition of piperine as an absorption promoter. The 13C CP MAS NMR spectra were recorded using variable contact time and dipolar dephasing experiment. The calculation of theoretical chemical shift values for three polymorphic forms of piperine allowed us to create a database of NMR parameters enabling the identification of polymorphic forms based on the analysis of the 13C CP MAS NMR spectrum. Additionally, the analysis of cross-polarization kinetics parameters was performed. Then, the 13C CP MAS NMR technique was used to confirm the authenticity and determine the presence of curcumin in dietary supplements containing curcumin with the addition of piperine. The presence of piperine could be confirmed even when the mass content of piperine was 70 times lower than that of curcumin. This method can be used to test the quality of dietary supplements containing the addition of piperine as an absorption promoter.

Searching for Natural Aurora a Kinase Inhibitors from Peppers Using Molecular Docking and Molecular Dynamics.

Natural products are the precursors of many medicinal substances. Peppers (Piper, Capsicum, Pimienta) are a rich source of compounds with potential multidirectional biological activity. One of the studied directions is antitumor activity. Little research has been carried out so far on the ability of the compounds contained in peppers to inhibit the activity of Aurora A kinase, the overexpression of which is characteristic of cancer development. In this study, molecular docking methods, as well as molecular dynamics, were used, looking for compounds that could inhibit the activity of Aurora A kinase and trying to determine whether there is a relationship between the stimulation of the TRPV1 receptor and the inhibition of Aurora A kinase. We compared our results with anticancer activity studied earlier on MCF-7 cell lines (breast cancer cells). Our research indicates that the compounds contained in peppers can inhibit Aurora A. Further in vitro research is planned to confirm the obtained results.

13C CPMAS NMR as an Alternative Method to Verify the Quality of Dietary Supplements Containing Curcumin.

Turmeric is a traditional Indian spice that has recently become very popular worldwide because it contains a powerful ingredient called curcumin, which has strong anti-inflammatory properties. Hence, dietary supplements containing extracts rich in curcumin have gained great popularity. The main problems related to curcumin-containing dietary supplements are poor water solubility and the fact that they are often faked by using synthetic curcumin instead of the plant extract. In this article, we propose the use of the 13C CPMAS NMR method to control the quality of dietary supplements. The analysis of 13C CPMAS NMR spectra supported by GIPAW computations allowed us to identify a polymorphic form present in dietary supplements (which affected the solubility of curcumin) and to point out a dietary supplement that could be faked by using synthetic curcumin. Further PXRD and HPLC investigations confirmed that the examined supplement contained synthetic curcumin instead of the genuine extract. Our method can be used for routine control, especially because the investigation is performed directly from the capsule/tablet content and does not require any special sample preparation.

Methods to Improve the Solubility of Curcumin from Turmeric.

Turmeric is a strong-taste component of spices characteristic of Indian cuisine. It is obtained from the turmeric rhizome (Curcumae longae rhizoma) and has been used for thousands of years not only for culinary purposes, but also for medicinal purposes. It contains a group of organic compounds called curcuminoids. Curcumin is the main representative of this group of compounds which is also most frequently studied. In recent years, bioactive curcuminoids (including curcumin in the first place) have become more and more popular due to a wide spectrum of their biological activity. The anticancer, antibacterial, anti-inflammatory, and antiaging effects of curcumin have been confirmed by numerous in vitro and in vivo studies, as well as in clinical trials. However, an obstacle to simple, clinical application of curcumin is its poor bioavailability (which is due to its hydrophobic nature) and its very weak water solubility. Therefore, many scientists are working on improving the solubility of curcumin in water, which is the topic of the present article. Attempts have been made to combine curcumin with nanoparticles (polysaccharide or silica). Nanosuspensions or complexes with cyclodextrins are also considered. A promising direction is the search for new polymorphic varieties as well as obtaining cocrystals with curcumin which are characterized by better water solubility.

Phytochemical Profile and Antioxidant Activity of Lavandula angustifolia and Lavandula x intermedia Cultivars Extracted with Different Methods.

Lavender is a valuable perennial plant from the Lamiaceae family. It is grown mainly for its essential oil, but it also contains polar bioactive compounds such as polyphenols and coumarins. Their level depends on the species, cultivars, geographical origin, climatic conditions, harvest time and extraction method. The authors investigated the effect of several extraction procedures (maceration, decoction and ultrasound-assisted extraction) applied to three cultivars of Lavandula angustifolia (Betty's Blue, Elizabeth, Hidcote) and two cultivars of Lavandula x intermedia (Grosso, Gros Bleu) on the yield of the polyphenolic compounds and antioxidant activity. HPLC analysis showed the presence of rosmarinic acid (2.52-10.82 mg/g), ferulic acid glucoside (2.94-8.67 mg/g), caffeic acid (1.70-3.10 mg/g), morin (1.02-13.63 mg/g), coumarin (1.01-5.97 mg/g) and herniarin (1.05-8.02 mg/g). The content of phenolic acids and flavonoids was higher in lavender, while the content of coumarins was higher in lavandin in all types of extracts. The antioxidant activity was determined by DPPH-EPR assay for antiradical properties (104.58-206.77 µmol Trolox/g) and FRAP assay for reducing properties (79.21-203.06 µmol Trolox/g). The obtained results showed that the cultivar is the dominant factor differentiating the samples. Still, the extraction method plays an important role in the final bioactive substances content and antioxidant properties of obtained extracts.

Qualitative and quantitative analysis of energy drinks using 1H NMR and HPLC methods.

Energy drinks (EDs) are widely consumed to stimulate psychomotor functions and improve the efficiency of the human body. They typically contain caffeine, taurine, sugars or sweeteners, vitamins and organic acids. EDs were selected for the study in terms of composition diversity, reflecting the variety of products available on the Polish market. The analysis of the composition of energy drinks was performed using 1H NMR spectroscopy, HPLC-DAD, titration and refractometric methods. Diagnostic signals in 1H NMR spectra of the citric acid, caffeine and niacinamide were used for quantitative analysis. The citric acid content in energy drinks ranged from 140 to 780 mg per 100 mL. The niacinamide content in the tested energy drinks varied from 3.4 to 9.7 mg per 100 mL and was usually higher than it was reported on the label. The amount of caffeine (from 19 to 40 mg) was slightly lower than labeled. Quantitative determinations by 1H NMR and HPLC are compatible and can be successfully used interchangeably. In the so-called "sugar-free" drinks, no simple sugars or sucrose were found, thus 1H NMR can easily and quickly recognize ED without the addition of sugar. Our studies showed that 1H NMR spectroscopy is a valuable tool for quality control of energy drinks.

Application of 1H NMR in the study of fatty acids composition of vegetable oils.

The fatty acid composition is a parameter that determines the quality and origin of vegetable oils. The standard method used in the analysis of fatty acid composition is gas chromatography (GC). In the last 20 years, however, the 1H NMR method has become more important in the analysis of fatty acids. Thanks to no need of special sample preparation, the high speed of analysis and the possibility to automate the process of analysis, 1H NMR is becoming a popular method of testing vegetable oils. It is possible to test oils both qualitatively and quantitatively, taking into account both the fatty acid profile and the level of minor components. In combination with statistical and chemometric methods, the analysis of 1H NMR spectra allows one to obtain much valuable information about the tested oil, considering its composition, quality, the presence of impurities, or the origin. The paper presents an overview of publications focusing on the application of the 1H NMR method in the profiling of fatty acids in vegetable oils.

1H NMR and chemometric methods in verification of hemp-seed oil quality.

Hemp oil is widely used in the food industry, and also in many other industries. It is characterized by a high content of unsaturated fatty acids with the optimal ratio of ω-6 to ω-3 fatty acids (3:1). Such composition means that this oil is often used in the food, pharmaceutical and cosmetic industries. Due to the popularity of hemp oil and its high content of unsaturated fatty acids, methods for the quick oil quality analysis are sought. Six samples of cold-pressed hemp oil were stored under different conditions (2-8 °C and 30 °C) for one month and then their 1H NMR spectra were recorded. The chemometric methods of PCA and OPLS-DA were used to perform the analysis of the spectra. The combination of those methods made it possible to observe the differences in the oil samples after a month of storage. Based on the 1H NMR spectra, it was possible to differentiate the time and conditions of the oil storage.

Phytochemical Profiling, Antioxidant and Anti-Inflammatory Activity of Plants Belonging to the Lavandula Genus.

Lavender is a valuable medicinal plant belonging to the Lamiaceae family. Currently 39 species are known, but only Lavandula angustifolia is a pharmacopoeial raw material. Lavender has a long history of medicinal use and mainly exhibits antioxidant, anti-inflammatory, sedative, antidepressant, spasmolytic, anticholinesterases, antifungal and antibacterial properties. Used internally, it relieves symptoms of mental stress and insomnia and supports digestion. Topical use of lavender in aromatherapy, neuralgia and antiseptics is also known. The constant interest in lavender, and in particular in Lavandula angustifolia, in the field of medicine and pharmacy is evidenced by the growing number of publications. In view of so many studies, it seems important to review traditional and modern extraction techniques that determine the chemical composition responsible for the antioxidant and anti-inflammatory effects of various extracts from the species of the Lavandula genus.

Spirostanol Sapogenins and Saponins from Convallaria majalis L. Structural Characterization by 2D NMR, Theoretical GIAO DFT Calculations and Molecular Modeling.

Two new spirostanol sapogenins (5ß-spirost-25(27)-en-1ß,2ß,3ß,5ß-tetrol 3 and its 25,27-dihydro derivative, (25S)-spirostan-1ß,2ß,3ß,5ß-tetrol 4) and four new saponins were isolated from the roots and rhizomes of Convallaria majalis L. together with known sapogenins (isolated from Liliaceae): 5ß-spirost-25(27)-en-1ß,3ß-diol 1, (25S)-spirostan-1ß,3ß-diol 2, 5ß-spirost-25(27)-en-1ß,3ß,4ß,5ß-tetrol 5, (25S)-spirostan-1ß,3ß,4ß,5ß-tetrol 6, 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 7 and (25S)-spirostan-1ß,2ß,3ß,4ß,5ß-pentol 8. New steroidal saponins were found to be pentahydroxy 5-O-glycosides; 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 5-O-ß-galactopyranoside 9, 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 5-O-ß-arabinonoside 11, 5ß-(25S)-spirostan-1ß,2ß,3ß,4ß,5ß-pentol 5-O-galactoside 10 and 5ß-(25S)-spirostan-1ß,2ß,3ß,4ß,5ß-pentol 5-O-arabinoside 12 were isolated for the first time. The structures of those compounds were determined by NMR spectroscopy, including 2D COSY, HMBC, HSQC, NOESY, ROESY experiments, theoretical calculations of shielding constants by GIAO DFT, and mass spectrometry (FAB/LSI HR MS). An attempt was made to test biological activity, particularly as potential chemotherapeutic agents, using in silico methods. A set of 12 compounds was docked to the PDB structures of HER2 receptor and tubulin. The results indicated that diols have a higher affinity to the analyzed targets than tetrols and pentols. Two compounds (25S)-spirosten-1ß,3ß-diol 1 and 5ß-spirost-25(27)-en-1ß,2ß,3ß,4ß,5ß-pentol 5-O-galactoside 9 were selected for further evaluation of biological activity.

Aronia melanocarpa Fruits as a Rich Dietary Source of Chlorogenic Acids and Anthocyanins: 1H-NMR, HPLC-DAD, and Chemometric Studies.

Aronia melanocarpa (Michx.) Elliott's (chokeberry) besides anthocyanins contains significant amounts of hydroxycinnamic acids: Chlorogenic and its isomer neochlorogenic acid. They exhibit antioxidant, anti-inflammatory, antidiabetic, and antibacterial activities, thus they can have a significant impact on the health-promoting properties of Aronia. The aim of our research was to determine the changes in the content of chlorogenic acids (CGAs) and anthocyanins during fruit development and ripening, with a particular emphasis on acids. Aronia fruit samples were collected from July to October on two organic farms in Poland. The chemical composition of the extracts was determined by NMR spectroscopy and HPLC-DAD. 1H-NMR and HPLC data were analyzed using chemometric analysis and multivariate statistics (PCA). The results showed that the content of chlorogenic acids and anthocyanins changes during ripening and depends on the time of harvest and the region of cultivation. A correlation between the time of CGAs reduction and the appearance of anthocyanins was also noticed. The result of our research was also a database in the form of NMR parameters, which allows analysis of the metabolite profile and tracking of its changes. The 1H-NMR spectrum showing anthocyanin and CGA resonance can be considered the Aronia berry fingerprint.

Geissospermiculatine, a New Alkaloid from Geissospermum reticulatum Bark.

A new alkaloid, geissospermiculatine was characterized in Geissospermum reticulatum A. H. Gentry bark (Apocynaceae). Here, following a simplified isolation protocol, the structure of the alkaloid was elucidated through GC-MS, LC-MS/MS, 1D, and 2D NMR (COSY, ROESY, HSQC, HMBC, 1H-15N HMBC). Cytotoxic properties were evaluated in vitro on malignant THP-1 cells, and the results demonstrated that the cytotoxicity of the alkaloid (30 µg/mL) was comparable with staurosporine (10 µM). Additionally, the toxicity was tested on zebrafish (Danio rerio) embryos in vivo by monitoring their development (0-72 h); toxicity was not evident at 30 µg/mL.

Structural studies of two capsaicinoids: dihydrocapsaicin and nonivamide. 13C and 15N MAS NMR supported by genetic algorithm and GIAO DFT calculations.

Capsaicinoids are alkaloid type capsaicin analogs with prospective pharmacological activity. However their solid state conformations have not been studied yet. As part of the study, cross polarization (CP) magic angle spinning (MAS) solid state 13C and 15N NMR spectra of dihydrocapsaicin (DHCAP) and nonivamide (NVA) were recorded. Solid state chemical shifts differ from their solution counterparts; remarkable differences occur for carbons C2', C6' and C7' linked to C1' in DHCAP and with methylene carbons C4-C8 in NVA. The doubling of some resonances in the spectra of solid NVA indicates that there are two molecules in the crystallographic asymmetric unit. DFT GIAO calculations of shielding constants were performed for several geometric isomers, including molecules with different orientations of aliphatic chain with respect to aromatic ring. Low-energy conformers were found by genetic algorithm methodology. Comparison of experimental 13C chemical shifts with theoretical (GIAO DFT) shielding parameters was helpful in predicting the most reliable geometry in the solid state. Cross polarization time constants T CP and relaxation times in the rotating frame T H 1ρ were obtained from variable-contact cross-polarization experiments. T H 1ρ are longer in the order: NVA < CAP < DHCAP.

Antioxidant, Cytotoxic, and Antiproliferative Activities and Total Polyphenol Contents of the Extracts of Geissospermum reticulatum Bark.

Geissospermum species are medically important plants due to their health-promoting effects. The objective of this study was to determine the antioxidant ability and antiproliferative and cytotoxic effects of infusions, tinctures, and ethanolic extracts of Geissospermum reticulatum barks in relation to the contents of total phenolics and flavonoids. Seven samples of barks were collected in various regions of Peruvian Amazonia. We found that the amount of total phenolics in the studied products varied from 212.40 ± 0.69 to 1253.92 ± 11.20 mg GAE/kg. In our study there is a correlation (R2 = 0.7947) between the results of antioxidants assays: FRAP and ORAC for tinctures, infusions, and ethanolic extracts of G. reticulatum barks. We have also observed antiproliferative activities of the ethanolic extracts on normal T-cells. These extracts have caused death on malignant cell lines (THP-1 and HL-60) and this data correlates well with their antioxidant capacity measured by ORAC method. Interestingly, the highest concentration of the ethanolic extract was not toxic in the zebrafish embryo developmental assay. Our results indicate that G. reticulatum is rich in antioxidants and have cytotoxic and antiproliferative properties. The data suggests potential immunosuppressive role of the extracts. This is the first study presenting the results of chemical and biological analysis of multiple preparations from G. reticulatum.

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate.

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP(+)/NBT/PMS (ß-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H37Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H37Ra PGI was 226 µg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H37Ra PGI inhibition by PEP.

Antigenotoxic, anti-photogenotoxic and antioxidant activities of natural naphthoquinone shikonin and acetylshikonin and Arnebia euchroma callus extracts evaluated by the umu-test and EPR method.

The aim of this study was to evaluate the antigenotoxic and antioxidant potential of shikonin (SH), acetylshikonin (ACS) and Arnebia euchroma callus extract (EXT). The antigenotoxic activity was investigated by the umu-test as the inhibition of the SOS system induction caused by genotoxic chemical agents - 4-nitroquinoline oxide and 2-aminoanthracene. Moreover the ability of SH, ACS and EXT to prevent photogenotoxicity triggered by chlorpromazine under UVA irradiation was measured. The cytotoxicity of EXT toward V79 Chinese hamster cell line was additionally assessed. Shikonin and acetylshikonin had no effect on 4-NQO induced genotoxicity whereas EXT demonstrated an unclear effect. The protection against 2AA induced genotoxicity was observed for all tested substances. The highest protection was demonstrated for EXT with inhibition of 66%. SH and ACS reduced 2AA genotoxicity with inhibition of about 60%. Under UVA the strongest and dose-dependent activity was observed for EXT. Acetylshikonin was a weak anti-photogenotoxin whereas shikonin had no clear effect. EXT was highly cytotoxic toward the V79 cell line - the cells' morphology was affected seriously and apoptosis was impacted. The antioxidant activity of SH, ACS and EXT was studied by means of electron paramagnetic resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. All three samples exhibited radical scavenging properties.

Solid-state NMR in the analysis of drugs and naturally occurring materials.

This article presents some of the solid-state NMR (SSNMR) techniques used in the pharmaceutical and biomedical research. Solid-state magic angle spinning (MAS) NMR provides structural information on powder amorphous solids for which single-crystal diffraction structures cannot be obtained. NMR is non-destructive; the powder sample may be used for further studies. Quantitative results can be obtained, although solid-state NMR spectra are not normally quantitative. As compared with other techniques, MAS NMR is insensitive and requires a significant amount of the powder sample (2-100mg) to fill the 1.3-7 mm ZrO2 rotor. This is its main drawback, since natural compounds isolated from plants, microorganisms or cell cultures are difficult to obtain in quantities higher than a few milligrams. Multinuclear MAS NMR routinely uses (1)H and (13)C nuclei, less frequently (15)N, (19)F, (31)P, (77)Se, (29)Si, (43)Ca or (23)Na. The article focuses on the pharmaceutical applications of SSNMR, the studies were aimed to control over manufacturing processes (e.g. crystallization and milling) investigation of chemical and physical stability of solid forms both as pure drug and in a formulated product. SSNMR is used in combination with some other analytical methods (DSC, XRD, FT-IR) and theoretical calculations of NMR parameters. Biologically active compounds, such as amino acids and small peptides, steroids and flavonoids were studied by SSNMR methods (part 4) providing valuable structural information. The SSNMR experiments performed on biopolymers and large natural products like proteins, cellulose and lipid layers are commented upon briefly in part 5.

¹H and ¹³C NMR-based sugar profiling with chemometric analysis and antioxidant activity of herbhoneys and honeys.

BACKGROUND: Herbhoneys, relatively new bee products, are expected to have interesting medicinal properties. However, there is still a lack of data concerning their composition and antioxidant properties. ¹H and ¹³C NMR spectroscopy coupled with chemometric analysis (PCA and PLS-DA) and antioxidant assays (DPPH-ESR and ORAC-FL) were used to study 25 samples of Polish herbhoneys and honeys. RESULTS: Antioxidant activity varied among the samples. The best properties were exhibited by cocoa and instant coffee herbhoneys. The contents of total polyphenols and total carotenoids in the studied samples were found to be 70-1340 mg GAE kg⁻¹ and 0-28.05 mg kg⁻¹ respectively. No significant differences between herbhoney and honey samples were found in their sugar profiles. The PCA of ¹³C NMR spectra of the samples in DMSO-d6 resulted in sample clustering due to sucrose content. CONCLUSION: Herbhoneys have similar antioxidant properties to traditional honeys, being therefore of equal nutritional value. There was a noticeable influence of the extract concentration on the observed antioxidant effect. For samples with high antioxidant activity, polyphenols were responsible for the observed effect. Sample clustering due to sucrose content in the NMR-PCA study allowed effortless detection of adulteration.