P2X7-Receptor Pathway Involvement in the Anti-Inflammatory Activity of Medicinal Plants.

Medicinal plants used in European folk medicine attached to Lamiales, Gentianales or Asterales orders are used to treat inflammatory disorders. Many targets have been identified but to date, implication of purinergic receptor P2X7 activation has not yet been investigated. We managed to evaluate the protective effect on P2X7 activation by plant extracts used as anti-inflammatory in European folk medicine by the YO-PRO-1 uptake dye in vitro bioassay. Results revealed that among our selected plants, species from Scrophularia and Plantago genus were able to decrease significantly P2X7 activation (>50 % at 0.1 and 1 µg/mL). UPLC/MS, dereplication and metabolomic analysis of Scrophularia extracts, allowed us to identify the cinnamoyl-iridoid harpagoside as putative inhibitor of P2X7 activation. These results open a new research field regarding the anti-inflammatory mechanism of cinnamoyl-iridoids bearing plants, which may involve the P2X7 receptor.

Bioassay-Guided Skin-Beneficial Effects of Fractionated Sideritis raeseri subsp. raeseri Extract.

This study aimed to evaluate the potentials for skin-beneficial effects of the hydroethanolic extract of the aerial parts of ironwort (Sideritis raeseri Boiss. & Heldr. subsp. raeseri) and its fractions (petroleum ether, ethyl acetate, n-butanol and water). For these purposes, chemical analysis and in vitro antioxidant, anti-tyrosinase and antimicrobial assays, as well as determination of sun protection factor (SPF), were performed. The highest total phenolic content and quantity of individual flavonoids, chlorogenic acid and verbascoside were determined in the n-butanol fraction, which is in line with results obtained for antioxidant activity. The greatest antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the most common causative agents of the skin infections in humans, was exhibited by the ethyl acetate fraction. The strongest anti-tyrosinase activity was shown by the hydroethanolic extract (52.64% of inhibition at 100 µg/mL). Almost all tested samples showed photoprotective activity with SPF higher than 6 obtained at a low concentration of 0.1 mg/mL, which was more than 15 for the butanol fraction. These findings revealed that the hydroethanolic extract of S. raeseri aerial parts could be a valuable ingredient in the formulation of cosmetic and medicinal products intended to overcome various skin disorders.

Ultrasound-Assisted Natural Deep Eutectic Solvents Extraction of Bilberry Anthocyanins: Optimization, Bioactivities, and Storage Stability.

Bilberry fruits (Vaccinium myrtillus L.) are one of the richest natural sources of anthocyanins and are widely used due to their pharmacological and nutritional properties. To ensure their maximum application potential, it is necessary to overcome the limitations of conventional extraction solvents and techniques. This study aimed to develop a green method for bilberry anthocyanin extraction using natural deep eutectic solvents (NaDES) integrated with ultrasound-assisted extraction (UAE) in order to define extraction conditions that will prevent decomposition of the anthocyanins or the loss of bioactivity. After a screening of ten different NaDES, choline chloride:sorbitol (1:1) was selected as the most effective. Furthermore, the influence analysis and optimization of the NaDES-UAE extraction conditions were carried out employing response surface methodology. The optimal conditions were found to be an extraction time of 37.63 min, a temperature of 48.38 °C, and 34.79% (w/w) water in NaDES. The extraction yields of target compounds under optimized extraction conditions were 0.27 mg/g DW of cyanidin-3-O-glucoside and 2.12 mg CGE/g DW of TAC. The obtained optimized extract showed promising radical scavenging and antimicrobial activity. A stability study with the optimized extract revealed that refrigerated storage at 4 °C in the dark provided the best anthocyanins preservation. Overall, the developed NaDES-UAE method showed promising application potential and can be considered as a high-efficiency green alternative to conventional anthocyanins extraction methods, enabling the preservation of active ingredients and the bioactivity of extracts.

Pomegranate peel extract polyphenols attenuate the SARS-CoV-2 S-glycoprotein binding ability to ACE2 Receptor: In silico and in vitro studies.

The novel coronavirus disease (Covid-19) has become a major health threat globally. The interaction of SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) with ACE2 receptor on host cells was recognized as the first step of virus infection and therefore as one of the primary targets for novel therapeutics. Pomegranate extracts are rich sources of bioactive polyphenols that were already recognized for their beneficial health effects. In this study, both in silico and in vitro methods were employed for evaluation of pomegranate peel extract (PoPEx), their major polyphenols, as well as their major metabolite urolithin A, to attenuate the contact of S-glycoprotein RBD and ACE2. Our results showed that PoPEx, punicalin, punicalagin and urolithin A exerted significant potential to block the S-glycoprotein-ACE2 contact. These in vitro results strongly confirm the in silico predictions and provide a valuable insight in the potential of pomegranate polyphenols for application in SARS-CoV-2 infection.

Optimization of Ultrasound-Assisted Extraction of Phenolics from Sideritis raeseri Using Response Surface Methodology.

In this study we define the optimal conditions for ultrasound-assisted extraction of bioactive polyphenols from S. raeseri aerial parts using response surface methodology. The influence of ethanol concentration (10-90%), extraction temperature (20-80 °C), extraction time (10-60 min), and solid-to-solvent ratio (1:10-1:50) on total phenolic content as well as on content of individual flavonoids, and hypolaetin and isoscutellarein derivatives was studied. For the experimental design, a central composite design was chosen. In the obtained extracts, the following ranges of targeted compounds were detected: total phenol from 19.32 to 47.23 mg GAE/g dw, HYP from 1.05 to 11.46 mg/g dw, ISC 1 from 0.68 to 10.68 mg/g dw, and ISC 2 from 0.74 to 15.56 mg/g dw. The optimal extraction conditions were set as: ethanol concentration of 65%, extraction time of 50 min, extraction temperature of 63 °C, and solid-to-solvent ratio of 1:40. Contents of TP, HYP, ISC 1, and ISC 2 in optimal extracts were 47.11 mg GAE/g dw, 11.73 mg/g dw, 9.54 mg/g dw, and 15.40 mg/g dw, respectively. Experimentally set values were in good agreement with those predicted by the response surface methodology model, indicating suitability of the used model, as well as the success of response surface methodology in optimizing the conditions of the extraction.

Antiviral Effects of Pomegranate Peel Extracts on Human Norovirus in Food Models and Simulated Gastrointestinal Fluids.

Human noroviruses (HuNoV) are the dominant cause of viral gastroenteritis in all age groups worldwide. In this study, we investigated the effects of pomegranate peel extract (PPE) on the reduction of HuNoV in different food models, on surfaces of fresh produce (green onion and cherry tomato), in low-fat milk, and simulated gastrointestinal fluids. The antiviral efficacy of PPE against HuNoV was evaluated by quantifying the number of residual virus genomes using a quantitative reverse transcription PCR (qRT-PCR) assay. Pomegranate peel, considered as a waste product of industrial processing, is known for beneficial health effects and broad antimicrobial activity due to the high content of phenolic compounds and tannins. PPE showed significant antiviral properties against HuNoV both in phosphate-buffered saline (PBS) and simulated gastric fluid. The reduction of HuNoV by pomegranate juice was lower than with PPE, which could be attributed to the lower content of antimicrobial compounds. A pretreatment of cherry tomato and green onion surfaces with PPE significantly reduced the amount of HuNoV particles that adhered to those surfaces during subsequent virus suspension treatment. A detrimental effect of PPE on HuNoV structure was confirmed by transmission electron microscopy. Our results indicate that PPE is a natural antiviral agent effective against food-borne noroviruses.

Mahonia aquifolium Extracts Promote Doxorubicin Effects against Lung Adenocarcinoma Cells In Vitro.

Mahonia aquifolium and its secondary metabolites have been shown to have anticancer potential. We performed MTT, scratch, and colony formation assays; analyzed cell cycle phase distribution and doxorubicin uptake and retention with flow cytometry; and detected alterations in the expression of genes involved in the formation of cell-cell interactions and migration using quantitative real-time PCR following treatment of lung adenocarcinoma cells with doxorubicin, M. aquifolium extracts, or their combination. MTT assay results suggested strong synergistic effects of the combined treatments, and their application led to an increase in cell numbers in the subG1 phase of the cell cycle. Both extracts were shown to prolong doxorubicin retention time in cancer cells, while the application of doxorubicin/extract combination led to a decrease in MMP9 expression. Furthermore, cells treated with doxorubicin/extract combinations were shown to have lower migratory and colony formation potentials than untreated cells or cells treated with doxorubicin alone. The obtained results suggest that nontoxic M. aquifolium extracts can enhance the activity of doxorubicin, thus potentially allowing the application of lower doxorubicin doses in vivo, which may decrease its toxic effects in normal tissues.

Comparative Study of Subcritical Water and Microwave-Assisted Extraction Techniques Impact on the Phenolic Compounds and 5-Hydroxymethylfurfural Content in Pomegranate Peel.

Two environmentally friendly innovative extraction techniques - subcritical water (SWE) and microwave-assisted extraction (MAE) were applied for the extraction of phenolics from pomegranate peel. The impact of process conditions (SWE: temperature 100-220 °C, extraction time 5-30 min; MAE: solvent water and 50% ethanol, irradiation power 470 and 800 W) on the quality of extracts in terms of the content of total phenolics, total flavonoids, major phenolic constituents (gallic acid, ellagic acid, punicalin, punicalagin), as well as 5-hydroxymethylfurfural(HMF) amount was investigated. For SWE, temperature of 130 °C and 20 min extraction time were found optimal for obtaining high content of bioactive compounds and minimizing the yield of HMF. During MAE, phenolic compounds were effectively extracted by using lower microwave power and 50% ethanol. Comparing two techniques, MAE is more efficient than SWE for the extraction of phenolics from pomegranate peel while obtaining a HMF-free extracts.

Traditional Use of Medicinal Plants in South-Eastern Serbia (Pcinja District): Ethnopharmacological Investigation on the Current Status and Comparison With Half a Century Old Data.

Balkan Peninsula is one of the most important biodiversity centers in Europe. Despite that, the usage of plant species in the traditional medicine of some Balkan regions remained largely unexplored in the past. This study aimed to collect and document data on the traditional use of medicinal plants in Pcinja district in South-Eastern Serbia, which is among the least developed regions in Serbia. Also, comparison with data collected by Dr. Jovan Tucakov, in a book called Herbal therapy was conducted. The survey was carried out using semi-structured interviews and 113 informants were interviewed. Quantitative ethnobotany factors were calculated, allowing us discussing the results. The informants reported data on 86 medicinal plants belonging to 43 families in Pcinja district. Lamiaceae, Asteraceae, and Rosaceae were the dominant locally used families. The species with the highest number of use reports were Mentha piperita, Matricaria chamomilla, and Hypericum perforatum. Gastrointestinal ailments, respiratory problems and skin diseases were the most frequently reported indications. Usually, the administration was primarily oral followed by topical applications. Leaves were dominantly exploited plant parts and the most frequent preparation form was infusion. Medicinal plants in Pcinja district are mainly used as a mode of primary health care for treating minor health issues. After comparing our results with the ones collected half a century ago by Dr. Jovan Tucakov we can conclude that plant species mentioned in our investigation previously had a much wider spectrum of application.

Effect of pomegranate peel extract on the oxidative stability of pomegranate seed oil.

Pomegranate seed oil is valuable source of bioactive compounds with health-beneficial effects, but it is sensitive to oxidation due to high content of PUFA. In order to improve stability of pomegranate seed oil, the influence of natural antioxidant (0.1% pomegranate peel extract) and a combination of synthetic antioxidant (0.01% butylhydroxytoluene) and pomegranate peel extract (0.05%) was investigated. The oxidation process was followed by monitoring fatty acid composition and measuring the quality and stability parameters of oil (acid value, peroxide value, anisidine value and thiobarbituric acid reactive substances value) during storage for 12 days at 65 °C. Furthermore, total phenolic content and DPPH radical scavenging activity was measured. Pomegranate peel extract as well as a combination of pomegranate peel extract and BHT can have a significant positive impact (P < 0.05) on improvement of the quality and stability parameters of pomegranate seed oil. Furthermore, such activity was better than BHT used alone.

Fucus spiralis extract and fractions: anticancer and pharmacological potentials.

PURPOSE: Sea macroalgae are an important source of biologically highly valuable compounds. The main aim of this study was to investigate the in vitro anticancer properties and chemical composition of the dichloromethane-methanol extract and three fractions of the Fucus spiralis from coastline of Morocco. METHODS: Fractions were made from dichloromethane:methanol (1:1) extract of Fucus spiralis: petroleum-ether, ethyl-acetate and n-butanol. Extract and fractions were screened for in vitro cytotoxicity by MTT assay against human cervical adenocarcinoma (HeLa), colorectal adenocarcinoma (LS-174T), lung carcinoma (A549), and normal human lung fibroblasts (MRC-5). Cell cycle distribution of the HeLa cells was evaluated using flow cytometry. Acridine orange (AO)-ethidium bromide (EB) staining was used to assess morphological changes of HeLa cells under fluorescence microscope. Anti-migration and anti-angiogenic properties were investigated using scratch and tube formation assays against human endothelium-derived permanent EA.hy926 cell line. Antidiabetic activity was tested using anti-α-glucosidase assay. Antimicrobial effect was tested using micro- dilution method. RESULTS: Petroleum-ether fraction оf Fucus spiralis rich in fatty acids exerted the highest cytotoxicity against HeLa cells. Ethyl-acetate and petroleum-ether fractions induced the highest accumulation of the HeLa cells in sub-G1 and G2/M phases. Extract and fractions showed proapoptotic effect on HeLa cells under fluorescent microscope. They exhibited antimigratory and antiangiogenic effects in vitro. IC50 value for α-glucosidase inhibitory activity was much stronger than standard acarbose. n-Butanol fraction exerted the highest antibacterial and antifungal activity. CONCLUSIONS: The investigation of various biological activities of the extract and fractions obtained from Fucus spiralis may suggest a promising anticancer and pharmacological potential of this edible macroalga.

Grape Seed Polyphenols and Fatty Acids of Autochthonous Prokupac Vine Variety from Serbia.

Grape pomace (grape skin, seeds, and stems) represents a valuable source of bioactive compounds, thus the interest in making use of these wine waste products is increasing. Prokupac is an autochthonous Serbian variety of black grape with a long tradition in production of red wine. Identification of the superficial polyphenolic compounds by LC/MS revealed presence of 19 compounds including hydroxybenzoic acid derivatives, ellagic acid derivatives, flavan-3-ol monomers, proanthocyanidins, and flavonols. Catechin and epicatechin together with proanthocyanidins were the most abundant compounds and the highest content of phenolic compounds was determined in Prokupac clones 43/1, 40/1, and 40/2. Grape seed oil consisted of dominantly UFA (87-95 %) where methyl linoleate was the most abundant in all samples (69-81 %). Palmitic (3-8 %) and stearic acid (2-4 %) methyl esters were dominant SFA. Prokupac clone 43/3 was the most abundant in UFA (95 %) with the lowest percentage of SFA (5 %), while clones 41/1 and 43/5 had the lowest percentage of UFA (87 %) and the highest content of SFA (13 %). Favorable chemical profile of Prokupac clone seeds implicates its potential to be used as a raw material in further processing and possibility to be included as one of the quality parameters in further selection of the most interesting Prokupac clone.

Identification of cytotoxic metabolites from Mahonia aquifolium using 1H NMR-based metabolomics approach.

Herein, we propose a 1H NMR-based metabolomics method to reveal cytotoxic metabolites from Mahonia aquifolium stem-bark. Primary and secondary metabolites in the Mahonia aquifolium extracts were identified by thorough analysis of 1H and 2D NMR spectra, without prior isolation. An OPLS multivariate analysis method was used to correlate the chemical composition of the plant extracts with the results of cytotoxic activity against Human cervical adenocarcinoma cell line. Protoberberine alkaloids berberinе and palmatine, along with bisbenzylisoquinoline alkaloid berbamine were identified as the most influential in the OPLS model, with the highest cytotoxic activity.

Sorbus aucuparia and Sorbus aria as a Source of Antioxidant Phenolics, Tocopherols, and Pigments.

Due to its nutritive and medicinal properties, berries of some Sorbus species are used for the preparation of jams and jelly as well as in traditional medicine. On the other hand, their chemical composition is not much studied especially of those grown in Balkan Peninsula. We have analyzed individual phenolics, tocopherols, carotenoids and chlorophylls using HPLC in berries from Sorbus aucuparia and Sorbus aria collected in different localities in Serbia and Montenegro together with the amounts of total phenolics and proanthocyanidins as well as their radical scavenging activity against DPPH radical. Berries of S. aucuparia were richer source of polyphenolics in comparision with S. aria and, regardless the species and locality, caffeoylquinic acids such as neochlorogenic and chlorogenic acid were the most abundant compounds. Among analyzed tocopherols the most abundant in all samples was α-tocopherol (0.48 - 19.85 µg/g dw) as it was ß-carotene among carotenoids (mean concentration of 0.98 µg/g dw in S. aucuparia and 0.40 µg/g dw in S. aria, respectively). Correlation between total phenolics and DPPH radical scavenging activity was noticed. Our study represents comprehensive report on chemical composition of S. aucuparia and S. aria which could contribute to a better understanding of their quality.

Correction: Rosa canina L.--new possibilities for an old medicinal herb.

Correction for 'Rosa canina L. - new possibilities for an old medicinal herb' by Jelena Zivkovic et al., Food Funct., 2015, DOI: 10.1039/c5fo00820d.

Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique.

Traditional maceration method was used for the extraction of polyphenols from chokeberry (Aronia melanocarpa) dried fruit, and the effects of several extraction parameters on the total phenolics and anthocyanins contents were studied. Various solvents, particle size, solid-solvent ratio and extraction time have been investigated as independent variables in two level factorial design. Among examined variables, time was not statistically important factor for the extraction of polyphenols. The optimal extraction conditions were maceration of 0.75mm size berries by 50% ethanol, with solid-solvent ratio of 1:20, and predicted values were 27.7mgGAE/g for total phenolics and 0.27% for total anthocyanins. Under selected conditions, the experimental total phenolics were 27.8mgGAE/g, and total anthocyanins were 0.27%, which is in agreement with the predicted values. In addition, a complementary quantitative analysis of individual phenolic compounds was performed using HPLC method. The study indicated that maceration was effective and simple technique for the extraction of bioactive compounds from chokeberry fruit.

Rosa canina L.--new possibilities for an old medicinal herb.

Health beneficial properties of Rosa canina species are mainly attributed to rose hips, while the leaves are usually discarded as waste. In the present study we investigated chemical constituents as well as antimicrobial and antibiofilm potential of R. canina methanolic leaf extract. Chemical analysis showed that dominant phenolic compounds are quercetin and isorhamnetin derivatives (isoquercetin and isorhamnetin-3-O-rutinoside). Among the tested bacteria, Pseudomonas aeruginosa and Salmonella typhimurium were the most susceptible to the activity of R. canina leaf extract with MIC and MBC values both 0.009 mg mL(-1). For most of the bacterial strains investigated the extract showed significantly higher activity compared to the used standard compounds streptomycin and ampicillin. Also the tested extract powerfully inhibited in vitro biofilm growth in Pseudomonas aeruginosa, Staphylococcus aureus, Leishmania monocytogenes and Escherichia coli at sub MIC-levels. With concentrations equal to 1/4 and 1/8 of MIC values biofilm growth of P. aeruginosa and E. coli was inhibited by 90%. The obtained results are significant for a wider and efficient use of R. canina leaves.

Chitosan crosslinked microparticles with encapsulated polyphenols: Water sorption and release properties.

Chitosan-glutaraldehyde microparticles were produced by emulsion crosslinking method to be used as drug delivery system for polyphenols from Thymus serpyllum L. aqueous extract. The effect of preparation conditions, chitosan concentration (1.5-3% w/v), and glutaraldehyde/chitosan (GA/Ch) mass ratio (0.15-1.20) on water and polyphenols transport properties was investigated. Swelling ratio of dry particles (68-230 µm) in water ranged from 280% to 530%, depending on the formulation. The decrease in swelling was observed with increased GA/Ch mass ratio (i.e. crosslinking degree) at the same chitosan concentration, or with increased chitosan concentration at the same GA/Ch mass ratio. The increase in GA/Ch mass ratio was also manifested by increased particle compactness i.e. decreased size and reduced surface roughness. The sorption capacity for polyphenols seems to be a complex interplay of swelling behaviour and interactions chitosan-glutaraldehyde-polyphenols identified by Fourier transmission infrared analysis. An increase in crystallinity of chitosan was observed upon crosslinking with glutaraldehyde and encapsulation of polyphenols, as observed by X-ray diffraction analysis. The results obtained from release kinetics of selected polyphenolic compounds (caffeic acid, rosmarinic acid, total flavonoids, and total phenol content) showed that polyphenols were released at a lower amount (2-4 times) in water, but more rapidly (45-120 min) in comparison with the release in gastric followed by intestinal simulated fluid (SGF-SIF) (120-240 min). The experimental results of the time-dependent swelling in water and polyphenols release in both, water and SGF-SIF, were analyzed with several mathematical models. The results depicted Fickian diffusion as the water transport mechanism. In the case of polyphenols, only empirical Weibull model could be suggested for describing release kinetics.

Biological and nutritional properties of blackcurrant berries (Ribes nigrum L.) under conditions of shading nets.

BACKGROUND: Changes of environmental factors, created under the influence of various shading nets, could significantly affect the biological characteristics of plants grown in such conditions as well as biosynthesis of primary metabolites and ascorbic acid. Five blackcurrant cultivars - 'Ben Sarek', 'Ben Nevis', 'Ben Lomond', 'Ometa' and 'Cacanska Crna' - were cultivated in the shade of two green polyethylene nets and exposed to direct sunlight during two experimental seasons. RESULTS: In the control treatment, all cultivars contained the highest amounts of soluble solid content and number of flower buds per shoot in both years of cultivation. The bushes exposed to direct sunlight had the highest sunburn damage to the berries and leaves, and loss of yield. The greatest yield loss caused by berry damage in the present study during the experimental period was in cultivar 'Ben Sarek': 9.0% in 2010 and 15.4% in 2011. Growing in the shade of light-green net contributed the highest content of ascorbic acid. Control bushes and those shaded by light-green net had significantly higher radical scavenging activity, with values between 1.15 and 1.22 mg mL(-1). CONCLUSION: Plants shaded by nets in both years of cultivation had lower damage to leaves and berries and percentage of loss of yield, and usage of the net would be economically advantageous to growers. Fruit of blackcurrant cultivars grown in shading conditions still represent a good source of valuable nutritive and biologically active compounds.

Beneficial Effects of Polyphenol-Rich Chokeberry Juice Consumption on Blood Pressure Level and Lipid Status in Hypertensive Subjects.

Epidemiological studies have shown a positive association between intake of foods rich in antioxidants and lower incidence of cardiovascular disease development. Polyphenols are considered the most abundant and important dietary antioxidants. The aim of this study was to evaluate effects of polyphenol-rich chokeberry juice consumption on 24-h ambulatory monitored blood pressure (BP) level in subjects with no pharmacologically treated high normal BP or grade I hypertension. Twenty-three subjects (12 men and 11 women) aged 33-67 were enrolled and instructed to consume 200 mL of juice daily for 4 weeks. Participants were divided in two groups, based on prevalence of sympathetic or parasympathetic activity. Measurements of biochemical parameters and heart rate variability analysis were also applied. At the end of the intervention period, average 24-h and awake systolic and diastolic BP were significantly decreased (P<.05). This was more pronounced in the group with prevalence of sympathetic activity. Significant reduction in triglyceride level (P<.05) and a reducing effect on total and low-density lipoprotein cholesterol were also found. Obtained results indicate a positive impact of regular chokeberry juice consumption on BP and lipid status in pharmacologically untreated hypertensive subjects.