The intraocular staining potential of anthocyanins and their retinal biocompatibility: a preclinical study

Purpose: To perform preclinical studies to determine the efficacy and safety of anthocyanins as stains for the internal limiting membrane (ILM) of the eye.

Materials and methods: Cyanidin (Cya), delphinidin (Del), luteolinidin (Lut), peonidin (Peo) and pelargonidin (Pel) were evaluated. These natural dyes were used to stain the lens capsule and ILM of pig eyes. The effects of these dyes on retinal cell viability was determined using a water-soluble tetrazolium salt assay, and oxidative stress was measured in vitro. Histopathology, in situ TUNEL labelling, transmission electronic microscopy (TEM), and electroretinography (ERG) were performed on rats following the intravitreal and subretinal injection of the neuroprotective dyes.

Results: All anthocyanins stained the lens capsule and ILM of the pigs at a concentration of 1?mg/ml. Del, Lut and Peo were non-toxic and produced survival rates in the ARPE19 and RGC5 cells that were similar to those in control cells. We treated eyes with H₂O₂ and three dyes (Del, Lut, and Peo) to explore the possible neuroprotective effects and observed significantly higher survival rates in the ARPE19 cells treated with Del, Lut or Peo and the RGC5 cells treated with Lut or Peo than those in the control cells. Three dyes were intravitreally and subretinally injected into rats in vivo, and the histology showed mildly disorganized retinal cell layers. TUNEL staining and TEM examinations did not reveal additional toxic effects. Rat ERGs were not altered after intravitreal injections.

Conclusions: This preclinical study, Del, Lut, and Peo show potential as staining agents and warrant further investigation as vital dyes.     

In vitro α-amylase and pancreatic lipase inhibitory activity of Cornus mas L. and Cornus alba L. fruit extracts

Less-common fruits from Cornus spp. (Cornaceae), also named dogwoods, have shown antidiabetic, antibacterial and anti-allergic properties and are thus considered a source of phytochemicals that are beneficial to human health. The study aimed to compare the chemical compositions of the aqueous and ethanolic extracts of lyophilized fresh-picked and commercially available dried fruits of Cornus mas (Cm, cornelian cherry) and Cornus alba (Ca) fruits using HPLC-DAD-MS/MS method. Simultaneously, the α-amylase and pancreatic lipase (PL) inhibitory activities of the prepared extracts were compared by in vitro fluorescence assay based on the kinetic hydrolysis of starch or oleate ester of 4-methylumbelliferone (MUO), respectively. Additionally, a bio-assay guided identification of compounds potentially responsible for the inhibition of pancreatic enzymes was performed. Iridoids (loganic acid, cornuside) and anthocyanins (pelargonidin 3-O-galactoside) were identified in the Cm fruit extracts. Flavonoids, such as quercetin and kaempferol derivatives, were detected in the Ca fruit extracts. The chromatographic separation of the constituents of Ca fruit provided a fraction containing phenolic acids derivatives, which inhibited PL activity by 69.9 ± 4.5% at a concentration of 7.5 μg·mL^?1. The IC₅₀ of hydroxytyrosol glucoside, isolated from the most active Ca fraction, was 0.99 ± 0.10 mg·mL^?1 indicating other constituents responsible for the fraction activity. The most active subfraction from Cm fruit (7.5 μg·mL^?1), which inhibited PL activity by 28.3 ± 1.5%, contained pelargonidin 3-O-galactoside. Loganic acid and cornuside in highly pure form did not inhibit lipase activity. The phytochemical constituents of Cm, and particularly of Ca fruit extracts, can inhibit pancreatic enzymes and thus might be considered effective preparations in the prevention and control of hyperlipidemia related diseases.     

Anthocyanin profile and antioxidant capacity of black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) from Cuevas Bajas,Spain

The present work deals with the study of the anthocyanin profile of two different black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) cultivars, associated with Antonina and Purple Haze varieties, from Cuevas Bajas (Málaga, Spain) and some of their antioxidant features. The main anthocyanins detected by LC–MS were found to correspond to five cyanidin-based anthocyanins: cyanidin 3-xylosylglucosylgalactoside, cyanidin 3-xylosylgalactoside and the sinapic, ferulic and coumaric acids derivative of cyanidin 3-xylosylglucosylgalactoside. The anthocyanins present in the black carrots were essentially acylated and their levels were found to correspond to 25% and 50% of the total phenolic content for the Purple Haze and Antonina varieties, respectively. Moreover, the reducing capacity of the two black carrots extracts (86.4 ± 8.0 and 182.0 ± 27 μM TE/100 g fw) and the radical scavenging ability (17.6 ± 9.0 and 240.0 ± 54.0 μM TE/100 g fw) expressed in Trolox equivalents units were determined. The antioxidant features of the black carrot extracts were shown to be significantly higher than those of orange carrots used herein for comparison. Overall, this work highlights the Cuevas Bajas black carrots as rich sources of anthocyanins with significant antioxidant capacities and good nutritional value.     

Stability and antioxidant activity of polyphenols in extracts of Myrtus communis L. berries used for the preparation of myrtle liqueur

Flavonoids and anthocyanins in berry extracts from Myrtus communis, prepared by following a typical Sardinia myrtle liqueur recipe, were identified by HPLC coupled with Electrospray Mass Spectrometry and quantified by HPLC coupled with Ultraviolet/Visible Detection in order to evaluate the stability of the extracts during 1 year of storage. Antioxidant activity was measured by using TEAC assay, and the free-radical scavenging activity was monitored during time of the stability evaluation.Anthocyanins have found to be the most instable compounds, but a considerable instability was observed also for flavonoids, suggesting the use of extracts not over 3 months from their preparation. The myrtle extract showed interesting free-adical scavenging activity. Antioxidant activity was preserved in 3 months.     

A natural alternative to sulphur dioxide for red wine production: Influence on colour, antioxidant activity and anthocyanin content

An alternate to sulphur dioxide natural antioxidant was tested during 2005 harvest on four different red vinifications, and was applied in each winemaking batch in combination with sulphur dioxide and on its own. Responses measured and analysed with uni- and multivariate statistics were: anthocyanin content, antioxidant capacity and classic oenological parameters. ANOVA revealed no significant effect of treatment in the antioxidant values of RA or diphenylpicrylhydrazyl (DPPH) of wines, but a paired t-test could differentiate wines treated only with SO₂ from those treated only with the novel product according to their RA values. Principal component analysis (PCA) of anthocyanin content of the treated wines gave five significant components, explaining 100% of variance and differentiating products treated only with SO₂ from those treated only with new product. PCA of oenological data explained 72% of variance in the first two components and wines were clearly differentiated on the basis of SO₂ or alternative treatment. In all cases, commercially acceptable red wines were produced, giving a possibility for partial substitution of SO₂ by a natural product.     

The effect of a water extract and anthocyanins of hibiscus sabdariffa L on paracetamol-induced hepatoxicity in rats

We investigated the effect of the water extract of the dried flowers of Hibiscus sabdariffa L. and Hibiscus anthocyanins (HAs) (which are a group of natural pigments occurring in the dried calyx of H. sabdariffa) on paracetamol-induced hepatotoxicity in rats. The water extract was given in lieu of drinking water for 2, 3 or 4 consecutive weeks, and the HAs were given orally at doses of 50, 100 and 200 mg/Kg for five consecutive days. Paracetamol was given orally at a dose of 700 mg/Kg to induce hepatotoxicity at the end of the water extract and Has treatments. Six hours thereafter the rats were killed and their liver function evaluated biochemically and histologically. Given for 4 weeks (but not for 2 or 3 weeks) the extract significantly improved some of the liver function tests evaluated, but did not alter the histology of the paracetamol-treated rats or the pentobarbitone-induced sleeping time. At a dose of 200 mg/Kg, the hepatic histology and the biochemical indices of liver damage were restored to normal. Lower does were ineffective. Pending more evaluation for safety and efficacy, the HAs can potentially be used in mitigating paracetamol-induced hepatotoxicity.     

Phenolic compounds and antioxidant activities of skins and seeds of five wild grapes and two hybrids native to Japan

Grape skins and seeds are sources of phenolic compounds that contribute to the sensory characteristics and beneficial bioactivity of wines and other processed foods. Hence, the study was aimed to evaluate and characterize the phenolic composition and antioxidant activities of five wild grapes native to Japan and two hybrids derived from the wild ones. Finally, the results were compared with those of two cultivated grapes (Vitis vinifera cv. Muscat of Alexandria, a white and V. labruscana cv. Bailey Alicante A, a tenturier-type grape). Among the skins of wild grapes, those of Shiohitashibudou (V. sp., an unidentified wild grape) contained the highest amount of total phenolics (13.8 mg g⁻¹ gallic acid equivalent of fresh weight) and antiradical activities (61.7 mmol g⁻¹ trolox equivalent of fresh weight). In contrast, Shiragabudou (V. shiragai) contained highest amount of seed total phenolics (16.5 mg g⁻¹ gallic acid equivalent of fresh weight). All wild grape seeds were rich in procyanidin monomers. The total phenolics and antiradical activities in skins of a hybrid grape Kadainou R-1 were higher than those of its parents. This study demonstrates that wild grapes are potential sources of neutraceutical phenolics and can thus be utilized as novel breeding resources.     

Red fruit juice quality and authenticity control by HPLC

The present work used International Federation of Fruit-Juice Producers (IFU) Method No. 71 with minor modifications for the analysis of anthocyanins, betacyanins, synthetic red pigments, hydroxycinnamic acids, hydroxybenzoic acids and catechins present in red fruit and vegetable juices and red-purple soft-drinks. The proposed HPLC method has been implemented with simultaneous UV-Visible and fluorescence detection and offers unambiguous composition results for 9 red fruit and vegetable juices: strawberry, red raspberry, blueberry, European cranberry, blackcurrant, sour cherry, red grape, purple carrot and purple prickly pear. Twenty-eight anthocyanins, 4 betacyanins, 1 natural and 6 synthetic pigments, 11 hydroxycinnamic acids, 6 hydroxybenzoic acids and 2 catechins were determined in a 30-min chromatogram. This method is useful for quality and authentication analyses of red fruit and vegetable juices, and red-purple soft-drinks. The use of a unique analysis method for polyphenol analysis is encouraged as a helpful tool to build up an unambiguous polyphenol composition database of foods.     

The effects of the anthocyanin-rich extract from red cabbage leaves on Allium cepa L. root tip cell ultrastructure

The effect of exogenously applied 250 μM anthocyanin-rich (ATH-rich) extract from red cabbage leaves on the ultrastructure of Allium cepa root meristematic cells was investigated. The tested extract slightly affected mitochondria, endoplasmic reticulum (ER), Golgi apparatus and vacuoles. In the presence of ATH, 62% of mitochondria converted to condensed type. In addition swollen, circular ER cisternae were sporadically observed. In the ATH-treated roots, one third of Golgi structures was characterized by the reduced number of vesicles. Moreover in 54% of vacuoles, the electron-dense granular and circular material appeared. Additionally, in the cytoplasm, the presence of numerous multivesicular bodies (MVB) was noticed.The observed ultrastructural modifications of mitochondria, and presumably also ER, probably resulted from the ability of an ATH to affect mitochondrial respiratory activity. The other changes in A. cepa root meristematic cell ultrastructure were connected with the transport of exogenously applied ATH into vacuoles. It seems that they are transported from the plasmolemma to the vacuole by multvesicular bodies (MVB), and there trapped by anthocyanic vacuolar inclusions (AVIs).However, none of the observed ultrastructural changes seemed to disturb cell functions, therefore the ATH-rich extract from red cabbage leaves may be regarded as cell-friendly and can be safely used as a detoxifying agent against heavy metal poisoning, as it is more and more often postulated.