Raluca Maria Pop

Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells

Blueberry consumption is associated with health benefits contributing to a reduced risk for cardiovascular disease, diabetes and cancer. The aim of this study was to determine the anthocyanin profile of blueberry extracts and to evaluate their effects on B16-F10 metastatic melanoma murine cells. Seven blueberry cultivars cultivated in Romania were used. The blueberry extracts were purified over an Amberlite XAD-7 resin and a Sephadex LH-20 column, in order to obtain the anthocyanin rich fractions (ARF). The antioxidant activity of the ARF of all cultivars was evaluated by ABTS, CUPRAC and ORAC assays. High performance liquid chromatography followed by electrospray ionization mass spectrometry (HPLC-ESI-MS) was used to identify and quantify individual anthocyanins. The anthocyanin content of tested cultivars ranged from 101.88 to 195.01 mg malvidin-3-glucoside/100g fresh weight. The anthocyanin rich-fraction obtained from cultivar Torro (ARF-T) was shown to have the highest anthocyanin content and antioxidant activity, and inhibited B16-F10 melanoma murine cells proliferation at concentrations higher than 500 μg/ml. In addition, ARF-T stimulated apoptosis and increased total LDH activity in metastatic B16-F10 melanoma murine cells. These results indicate that the anthocyanins from blueberry cultivar could be used as a chemopreventive or adjuvant treatment for metastasis control.

UHPLC/PDA–ESI/MS Analysis of the Main Berry and Leaf Flavonol Glycosides from Different Carpathian Hippophaë rhamnoides L. Varieties

INTRODUCTION Sea buckthorn (Hippophaë rhamnoides L.) is known to be rich in many bioactive compounds (such as vitamins, phenolics, carotenoids) important for human health and nutrition. Among the phenolics, berries and leaves contain a wide range of flavonols that are good quality and authenticity biomarkers. OBJECTIVE To compare the composition of the main flavonols of Romanian sea buckthorn berry and leaf varieties and to identify the specific biomarkers that contribute to sample differentiation among varieties. MATERIAL AND METHODS Six varieties of cultivated sea buckthorn (ssp. Carpatica) berries and leaves were analysed by UHPLC/PDA-ESI/MS. RESULTS Berries and leaves contained mainly isorhamnetin (I) glycosides in different ratios. Whereas I-3-neohesperidoside, I-3-glucoside, I-3-rhamnosylglucoside, I-3-sophoroside-7-rhamnoside and free isorhamnetin were predominant for berries (out of 17 compounds identified), I-3-rhamnosylglucoside, I-3-neohesperidoside, I-3-glucoside, quercetin-3-pentoside, kaempferol-3-rutinoside, and quercetin-3-glucoside were predominant in leaves (out of 19 compounds identified). Berries contained, on average, 917 mg/100 g DW flavonol glycosides. Leaves had higher content of flavonol glycosides than berries, on average 1118 mg/100 g DW. The variation of the quantitative dataset analysed using principal component analysis accounted for 91% of the total variance in the case of berries and 73% in case of leaves, demonstrating a good discrimination among samples. CONCLUSION Based on quantitative analysis, by principal component analysis, the flavonol derivatives can be considered as biomarkers to discriminate among varieties and to recognise specifically the berry versus leaf composition.

Determination of Carotenoids and their Esters in Fruits of Sea Buckthorn (Hippophae rhamnoides L.) by HPLC-DAD-APCI-MS

INTRODUCTION The berries of Hippophae rhamnoides Linnaeus have high nutritional and medicinal values and have been used for centuries as food both in Europe and Asia. The oleoresins represent a potential source of carotenoid esters and can be used as food additives, cosmetic ingredients or nutraceuticals. OBJECTIVE The objective of this study was to develop a HPLC-DAD-APCI-MS method, with both positive and negative ionisation modes, for the direct identification of the native carotenoid composition in fruits of Hippophae rhamnoides. MATERIALS AND METHODS Fruits of Hippophae rhamnoides, cv. Serbanesti and Victoria, were collected from an experimental field at the Fruit Research Station of Bacau, Romania. Samples were extracted using methanol:ethyl acetate:petroleum ether (1:1:1, v/v/v). The HPLC-DAD-APCI-MS analyses were carried out on a Shimadzu system using a YMC C₃₀-column and a gradient elution. RESULTS In total 22 compounds were detected, eight were free carotenoids, nine were xanthophylls monoesters and five were xanthophylls diesters. Differences were observed in the relative percentage composition of the identified components among the two cultivars investigated. Zeaxanthin-C16:0,C16:0 was the most abundant diester. The unsaturated palmitoleic acid was directly detected in its esterified form, in zeaxanthin-C16:0,C16:1, which is reported here for the first time. Although present in small amounts the unsaturated oleic, linoleic, linolenic, hexadecadienoic and hexadecatrienoic acids were detected in their esterified forms as lutein monoesters, the last two having been detected in Hippophae rhamnoides for the first time. CONCLUSION A novel (HPLC-DAD-APCI-MS) method was developed for the direct identification of the native carotenoid composition in fruits of Hippophae rhamnoides.

Sodiation as a tool for enhancing the diagnostic value of MALDI-TOF/TOF-MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono- and diesters. For rapid fingerprinting of these esters, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono- and diester palmitate standards in MALDI-TOF/TOF-MS showed that sodium adduct parent masses [M + Na](+) gave much simpler MS(2) spectra than radical / protonated [M](+●) / [M + H](+) parents. [M + Na](+) fragments yielded diagnostic polyene-specific eliminations and fatty acid neutral losses, whereas [M](+●) / [M + H](+) fragmentation resulted in a multitude of non-diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na](+) ionization by addition of sodium acetate, and best signal-to-noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI-TOF/TOF-MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono- and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all-trans esterified esters found in LC were identified with MALDI-TOF/TOF-MS, with the exception of two minor monoesters.

Carotenoid and fatty acid profiles of bilberries and cultivated blueberries from Romania

The worldwide consumption of blueberries has increased due to their potential health effects. They contain micronutrients, various phytochemicals and anti-oxidant pigments, represented especially by anthocyanins. In addition to these, carotenoids and fatty acids are also present. The aim of this study was the identification and quantification of carotenoids (by HPLC) and fatty acids (by GC with flame ionisation detection) in two bilberry genotypes (Wild 1 and 2) and three cultivars (BlueCrop, Elliot, and Duke). The main carotenoids identified were lutein, β-cryptoxanthin, and β-carotene, with the average total carotenoid content of 266 μg per 100 g of fruit. The ratio of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs) was 0.098 for Wild 1 and 0.11 for Wild 2 genotypes and ranged from 0.14 to 0.28 for cultivars. The main fatty acids identified were linoleic (18:2) and linolenic (18:3) acids in all the samples analysed. In Vaccinium myrtillus the amount of carotenoids and fatty acids was higher than in Vaccinium corymbosum species.

Berberine: Botanical Occurrence, Traditional Uses, Extraction Methods, and Relevance in Cardiovascular, Metabolic, Hepatic, and Renal Disorders

Berberine-containing plants have been traditionally used in different parts of the world for the treatment of inflammatory disorders, skin diseases, wound healing, reducing fevers, affections of eyes, treatment of tumors, digestive and respiratory diseases, and microbial pathologies. The physico-chemical properties of berberine contribute to the high diversity of extraction and detection methods. Considering its particularities this review describes various methods mentioned in the literature so far with reference to the most important factors influencing berberine extraction. Further, the common separation and detection methods like thin layer chromatography, high performance liquid chromatography, and mass spectrometry are discussed in order to give a complex overview of the existing methods. Additionally, many clinical and experimental studies suggest that berberine has several pharmacological properties, such as immunomodulatory, antioxidative, cardioprotective, hepatoprotective, and renoprotective effects. This review summarizes the main information about botanical occurrence, traditional uses, extraction methods, and pharmacological effects of berberine and berberine-containing plants.