Neda M. Mimica-Dukić

Plantain (Plantago L.) Species as Novel Sources of Flavonoid Antioxidants

To examine the antioxidant properties of methanol extracts of selected Plantago species (P. argentea Chaix., P. holosteum Scop., P. major L., P. maritima L., and P. media L.), various assays that measure free radical scavenging ability were carried out: DPPH, hydroxyl radical, superoxide anion, and nitric oxide scavenger capacity tests, reducing power (FRAP) assay, and Fe(2+)/ascorbate induced lipid peroxidation. In all of the tests extracts showed a potent antioxidant effect compared with BHT, a well-known synthetic antioxidant, and the extract of P. major, accepted as an official remedy. Besides, in examined extracts the total phenolic amount (ranging from 38.43 to 70.97 mg of GAE/g of dw) and the total flavonoid content (5.31-13.10 mg of QE/g of dw) were determined. Furthermore, the presence and content of selected flavonoids (luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin, rutin, and quercetin) were studied using LC-MS/MS technique. LC-MS/MS analysis showed noticeable qualitative and quantitative differences between the species according to which the examined Plantago species could be regarded as a possible new source of natural antioxidants. In this study three of the species examined, P. maritima, P. argentea, and P. holosteum, have been analyzed for the first time.

Development of a rapid resolution HPLC method for the separation and determination of 17 phenolic compounds in crude plant extracts

AbstractRapid Resolution HPLC/DAD method, on a 1.8 µm, 4.6×50 mm column, was developed to enable a rapid separation of a mixture of 17 compounds, which consisted of hydroxybenzoic acids, hydroxycinnamic acids, flavones, flavonols, flavanone, flavonol-glycoside and antraquinone, in a single run, within 22 minutes. The developed method is precise, accurate and sensitive enough for simultaneous quantitative evaluation of major compounds in crude and hydrolyzed extracts of parsley, buckthorn, mint, caraway and birch. In order to overcome the inability to quantify all the phenolic compounds present in the samples caused by lack of external standards, HPLC approaches for the total phenolic content estimation based on sum of all integrated peak areas were made. These results were compared with the total phenolic content determined by Folin-Cioacalteu method. Although the correlation between the series of data was not significant (p<0.05), the difference between the results of total phenolic content obtained spectrophotometrically and by HPLC was not high in the case of parsley, buckthorn and mint extract. Regarding the obtained results, the HPLC approach could serve as an excellent tool for total phenolic content estimation, without the need for complete identification of the individual compounds.

Antioxidant activity relationship of phenolic compounds in Hypericum perforatum L.

BackgroundThe St Johns Wort (Hypericum perforatum; Clusiaceae) has been used in traditional and modern medicine for a long time due to its high content of biologically active phenolics. The purpose of this work was to develop a method for their fractionation and identification, and to determine the most active antioxidant compounds in plant extract.ResultsAn LC-MS method which enables fast qualitative and semiquantitative analysis was developed. The composition determined is in agreement with the previous results, where 6 flavonoids, 4 naphthodianthrones and 4 phloroglucinols have been identified. Significant antioxidant activity was determined for most of the fractions by DPPH assay (the lowest IC50 of 0.52 μg/ml), NO scavenging (6.11 μg/ml), superoxide scavenging (1.86 μg/ml), lipid peroxidation (0.0079 μg/ml) and FRAP (the highest reduction capacity of 104 mg Fe equivalents/g) assays.ConclusionLC-MS technique has been successfully applied for a quick separation and identification of the major components of H. perforatum fractions. Majority of the fractions analyzed have expressed a very high antioxidative activity when compared to synthetic antioxidants. The antioxidant activity could be attributed to flavonoids and phenolic acids, while phloroglucinols and naphthodianthrones showed no significant activity. It is demonstrated that it is possible to obtain, by fractionation, H. perforatum preparations with significantly increased phloroglucinols-to-naphthodianthrones ratio (up to 95:5).

Liquid chromatography/tandem mass spectrometry study of anti-inflammatory activity of plantain (Plantago L.) species.

To evaluate anti-inflammatory activity of selected Plantago species (P. lanceolata L. and P. major L.) an optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken. By using intact cell system (platelets) as a source of COX-1 and 12-LOX enzymes and highly sensitive and specific LC-MS/MS technique for detection of main arachidonic acid metabolites formed by COX-1 and 12-LOX, this test provides efficient method for evaluation of anti-inflammatory potential of plant extracts and isolated compounds. Our results validated the well-known COX-1 inhibitory activity of P. lanceolata and P. major methanol extracts (concentration required for 50% inhibition (IC(50)) was 2.00 and 0.65 mg/ml, respectively). Furthermore, 12-LOX inhibitory activity of examined extracts was reported for the first time (IC(50)=0.75 and 1.73 mg/ml for P. lanceolata and P. major, respectively). Although renowned inhibitors, such as acetylsalicylic acid and quercetin showed higher activity, this study verifies P. lanceolata and P. major as considerable anti-inflammatory agents.

Antioxidant properties of Galium verum L. (Rubiaceae) extracts

The antioxidant properties of methanol extracts of Lady’s Bedstraw (Galium verum L., Rubiaceae) herb from two different localities in Serbia were evaluated. Antioxidant activity was assessed in four different model systems. Free radical scavenging capacity (RSC) was examined by measuring the scavenging activity of extracts on 2,2-diphenyl-1-pycrylhydrazil (DPPH) and hydroxyl radical (OH), as well as on hydrogen peroxide. In addition, the protective effects of lipid peroxidation (LP) in corn oil were evaluated by the TBA-assay using the Fe2+/ascorbate system of induction. The amount of dried extract, the content of total phenolics, flavonoids and chlorophylls was also determined. Extracts from both locations expressed very strong scavenger activity, reducing the DPPH⊙ (IC50=3.10 µg/mland 8.04 µg/ml) and OH radical formation (IC50=0.05 µg/ml and 0.54 µg/ml) and neutralising H2O2 (IC50=4.98 µg/ml and 3.80 µg/ml), in a dose dependant manner. Also, examined extracts showed notable inhibition of LP (IC50=11.69 µg/ml and 19.47 µg/ml). The observed differences in antioxidant activity could be partially explained by the levels of phenolics (2.44–4.65 mg and 4.57–5.16 mg gallic acid equivalents/g dry extract), flavonoids (6.38–10.70 µg and 15.56–17.96 µg quercetin equivalents/g dry extract) and chlorophylls in the investigated Lady’s Bedstraw extracts.

Comparative study of biological activities and phytochemical composition of two rose hips and their preserves: Rosa canina L. and Rosa arvensis Huds.

The aim of this study was to compare phenolic profile, vitamin C content, antioxidant, anti-inflammatory and cytotoxic activity of rose hips and the preserves (purée and jam) of two Rosa species: renowned Rosa canina L. and unexplored Rosa arvensis Huds. The liquid chromatography-tandem mass spectrometry analysis of 45 phenolics resulted in quantification of 14 compounds, with quercitrin, gallic and protocatechuic acids as the most dominant. High antioxidant potential of R. canina and a moderate activity of R. arvensis extracts were determined through several assays. Purée of both species and methanol extract of air-dried R. canina hips showed some anti-inflammatory (cyclooxygenase-1 and 12-lipooxygense inhibition potency) activity. Purée of R. canina exerted cytotoxic activity only against the HeLa cell line among several others (HeLa, MCF7, HT-29 and MRC-5). The presented results support traditional use of rose hips and their fruit preserves as food with health and nutritional benefits.

Phenolic profile, antioxidant, anti-inflammatory and cytotoxic activities of black (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles

The aim of this study was a comprehensive investigation on phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of the still insufficiently explored black summer truffles (Tuber aestivum Vittad.) and white (Tuber magnatum Pico) truffles. Methanol and water extracts, obtained by maceration and Soxhlet extraction, were examined. Forty-five phenolics was studied using LC-MS/MS: presence of 14 compounds was confirmed, with the most dominant being p-hydroxybenzoic acid, baicalein and kaempferol (T. aestivum), epicatechin and catechin (T. magnatum). Moderate antioxidant activity of both species was determined through several assays. Only T. magnatum showed anti-inflammatory potential by inhibiting COX-1 and 12-LOX pathway products synthesis. Methanol extracts exerted cytotoxicity against some tumour cell lines (HeLa, MCF7, HT-29), besides the prominent activity of water extracts towards breast adenocarcinoma (MCF7). To conclude, these results support further investigations of phytochemicals and biological activity towards verification of nutraceutical use of both species.

Lignicolous fungi from northern Serbia as natural sources of antioxidants

As a result of an interest in natural derived metabolites, lignicolous fungi have taken on great importance in biochemical investigations. In the present study, antioxidative screening analyses have included in vitro testing of different extracts (aqueous, methanol, chloroform) of four fungal species using three different assays: Fe2+/ascorbate-induced lipid peroxidation by TBA assay, the neutralisation of OH· radicals and the radical scavenging capacity with the DPPHk]assay. TLC analysis confirmed the existance of phenolics in the extracts, but also indicates the presence of some other compounds. The obtained results indicate that MeOH extracts manifested a degree of activity higher than that of CHCl3 extracts. With respect to antioxidative activity, the extracts can be ranged in the following declining order: G. lucidum, G. applanatum, M. giganteus and F. velutipes. These results suggest that analyzed fungi are of potential interest as sources of strong natural antioxidants that could be used in the food industries and nutrition.

Phytochemical composition and in vitro functional properties of three wild rose hips and their traditional preserves

The aim of the present study was investigation of the phenolic profile, ascorbic acid content, antioxidant, anti-acetylcholinesterase, anti-inflammatory and cytotoxic activity of rose hips and the preserves (purée and jam) of three insufficiently examined Rosa species: Rosa dumalis Bechst., R. dumetorum Thuill. and R. sempervirens L. The liquid chromatography-tandem mass spectrometry analysis resulted in quantification of 14 of the 45 phenolic compounds examined, with ellagic acid as the most dominant. Notable antioxidant activity of all three species was confirmed through several assays. Moderate inhibition of acetylcholinesterase by extracts of all investigated Rosa species was observed. Several extracts of examined Rosa species demonstrated inhibition potency towards production of some monitored eicosanoids in cyclooxygenase-1 and 12-lipoxygenase pathways. Two R. sempervirens extracts exerted cytotoxic activity against HeLa and HT-29 cell lines, but were inactive towards MRC-5 and MCF7. The results support the potential of these rose hips as food with health-promoting properties.

Polyphenolic profile, antioxidant and neuroprotective potency of grape juices and wines from Fruška Gora region (Serbia)

ABSTRACT This article reports novel and valuable data about three red (Cabernet Sauvignon, Frankovka and Merlot varieties), one rosé (Muscat Hamburg) and three white wines (Italian Riesling, Chardonnay and Sila), together with their related products (grape juices and young wines) produced in Fruška Gora vineyards, Serbia, concerning their in-depth polyphenolic profile and biological activities (antioxidant and neuroprotective). LC-MS/MS analysis of 47 phenolics showed that the phenolic acids, particularly ellagic (up to 8.99 mg/L, Cabernet Sauvignon) and caffeic acids (up to 2.83 mg/L, Frankovka) were generally more abundant in red wines. Also, the content of catechin and epicatechin (0.32–7.70 and 0.4–15.5 mg/L) as the leading flavonoids, malvidin 3-O-glucoside as the dominant anthocyanin (2.7–160 mg/L) or stilbene resveratrol (0.06–2.42 mg/L), were also higher in the red grape products. Overall antioxidant dominance of the red grape products, Merlot variety being the main one, was confirmed through several assays (DPPH• and •NO scavenging ability, reducing potential and inhibition of lipid peroxidation). In addition, grape juices and young wines, which are a good source of dietary natural antioxidants, were also evidenced. Furthermore, the optimised in vitro acetylcholinesterase inhibition assay was applied, indicating the grape juices and red wines as the most promising neuroprotective agents. Abbreviations: AAE: ascorbic acid equivalents; AChE: acetylcholinesterase; BHT: butylated hydroxytoluene; DPPH: 2,2-diphenyl-1-picrylhydrazyl; dw: dry weight; FA: fatty acids; FRAP: Ferric Reducing Antioxidant Power; LP: lipid peroxidation; MDA: malondialdehyde; SRM: selected reactions monitoring; PCA: principal component analysis; PG: propyl gallate; TBA: 2-thiobarbituric acid; TPTZ: 2,4,6-tripyridil-s-triazine.