Determination of Anthocyanins, Total Phenolic Content, and Antioxidant Activity in Andes Berry (Rubus glaucus Benth)

ABSTRACT:  Anthocyanins, total phenolic content, ascorbic acid content, and the antioxidant activity were determined in extracts of Andes berry fruit ( Rubus glaucus Benth). Anthocyanis (ACNs) were isolated and characterized by means of high-performance liquid chromatography (HPLC) with photodiode array detection and electro spray ionization/mass spectrometry (PDA-ESI/MS/MS) analysis. The anthocyanin (ACN) content was 45 mg/100 g FW. The isolated anthocyanins were characterized as cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylorutinoside, cyanidin 3-rutinoside, pelargonidin 3-glucoside, and pelargonidin 3-rutinoside. The ascorbic acid content was 10.1 mg/100 g FW. The total phenolic content as determined by the Folin–Ciocalteau method was 294 mg GAE/100 g FW while the antioxidant activity as measured by ABTS ^{· +} radical scavenging capacity and ferric reducing antioxidant power (FRAP) was 2.01 and 4.50 mmol TE/100 g FW or 8.22 mmoles ferric iron reduced/100 g FW, respectively. The high phenolic content and antioxidant capacity of Andes berry suggest that this fruit could be a rich source of natural pigments, nutraceuticals, and natural antioxidants.     

Nutritional composition of commercial pickled garlic

The effect of pulp treatment on the qualitative and quantitative changes to polyphenol compounds, such as anthocyanins and flavanols, in musts and wines from blackcurrant and cherries was investigated. The following variants of pulp treatment were used: hot maceration, hot maceration and pulp pectinolysis with Rohapect MA Plus and Pectopol PM preparations, and also pulp pectinolysis with Rohapect and Pectopol preparations. The method of treatment affected the content of anthocyanins and flavan-3-ols in the musts. Different types of phenolic compounds reacted differently under must-making conditions. Wines made with different treatments presented statistical differences with the control wine and between them for the parameters studied.As a result of the analysis of cherry musts and wines by HPLC, the following flavan-3-ols were identified: catechin, epicatechin, dimer B₂ and trimer C₁. In the cherry wines studied, in the variants subjected to pectinolysis and fermentation in the pulp, epicatechin occurred in a smaller amount than catechin, while in the wines subjected to thermal treatment it was predominant. In the blackcurrant musts and wines the following flavanols were identified: gallocatechin, catechin, epigallocatechin, dimer B₂, epicatechin and trimer C₁. In the cherry musts and wines the following anthocyanin pigments have been identified: cyanidin 3-glucoside, cyanidin 3-rutinoside and cyanidin 3-glucosylrutinoside, the amount of which was the greatest. Anthocyanins identified in the blackcurrant musts and wines were delphinidine and cyanidine glycosides: delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-glucoside and cyanidin 3-rutinoside; their aglycones were also found.     

ANTHOCYANIN PIGMENTS OF SOUR CHERRIES

Fresh fruits of tart cherries (Prunus cerasus L. var. Montmorency) were extracted with acidic methanol. The anthocyanin pigments were isolated and purified by conventional paper chromatography. They were identified by spectral and R_f data and by acid hydrolysis. The pigments were cy anidin-3-glucosylrutinoside, cyanidin-3-glucosylsambubioside, cyani-din-3-sophoroside, cyanidin-3-rutinoside, cyanidin-3-glucoside and peoni-din-3-rutinoside. No free cyanidin or peonidin was found. Cyanidin-3-glucosylsambubioside, reported in cherries for the first time, was also found in the varieties of English Morello, Early Richmond and Meteor     

Berry anthocyanins: isolation,identification and antioxidant activities

Anthocyanins from bilberry, blackcurrant and cowberry were isolated for antioxidant evaluation. Individual compounds were identified and quantified using HPLC and HPLC/ESI–MS techniques. Antioxidant and radical‐scavenging capacities of the isolates were studied in emulsified methyl linoleate and human low‐density lipoprotein (LDL) in vitro and in the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) test. The total anthocyanin contents in the phenolic extracts of bilberry, blackcurrant and cowberry were 6000, 2360 and 680 mg kg^?1 fresh weight respectively. There were four dominant compounds in blackcurrant (glucosides and rutinosides of cyanidin and delphinidin), three in cowberry (monoglycosides of cyanidin) and 15 in bilberry (monoglycosides of cyanidin, delphinidin, malvidin, peonidin and petunidin). Quantification as cyanidin‐3‐glucoside equivalents gave markedly lower results regarding the total anthocyanin concentration and the content of individual delphinidin and malvidin compounds compared with quantification based on corresponding standard compounds. Berry anthocyanins were highly active radical scavengers in the DPPH test and effective antioxidants in emulsion and human LDL. Copyright © 2003 Society of Chemical Industry     

Evolution of juice anthocyanins during ripening of new selected pomegranate (Punica granatum) clones

 For five new clones of pomegranate, cultivated under homogeneous conditions, changes in juice anthocyanin contents during ripening were studied. Six anthocyanin pigments were found to be responsible for the red color of pomegranate juice. These were quantitatively and qualitatively analyzed by high-performance liquid chromatography and identified as delphinidin 3-glucoside and 3,5-diglucoside, cyanidin 3-glucoside and 3,5-diglucoside and pelargonidin 3-glucoside and 3,5-diglucoside. Generally, there was an increase in juice pigmentation during fruit ripening. In the early fruit-ripening stages, delphinidin 3,5-diglucoside was the main pigment, followed by cyanidin 3,5-diglucoside, while in the later stages, the monoglucoside derivatives cyanidin 3-glucoside and delphinidin 3-glucoside increased considerably. The pelargonidin derivatives were always present in small amounts. Received: 1 March 1999     

Anthocyanin and colour changes during processing of pomegranate (Punica granatum L., cv. Hicaznar) juice from sacs and whole fruit

The effects of clarification and pasteurisation on anthocyanins (ACNs) and the colour of pomegranate juice (PJ) produced from sacs and whole fruits were investigated. Clarification caused a loss of 4% of ACNs in juice from sacs (JFS) and a loss of 19% in juice from whole fruit (JFWF). After pasteurisation, there was an 8–14% and 13–9% loss of ACNs from unclarified and clarified JFS and JFWF samples, respectively. Polymeric colour was very high even in unclarified samples (25–29%). Compared to JFS, higher polymeric colour was formed in JFWF. HPLC analyses of PJ revealed that cyanidin-3,5-diglucoside was the major ACN, followed by cyanidin-3-glucoside and delphinidin-3-glucoside. Cyanidin-3,5-diglucoside showed higher stability to clarification and pasteurisation than cyanidin-3-glucoside in both PJ samples. Cold clarification with only gelatin is recommended for PJ. To prevent excessive ACN loss and the formation of brown colouring, PJ should be subjected to minimal heating.     

Anthocyanin Determination in Black Raspberry (Rubus occidentalis) and Biological Specimens Using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

ABSTRACT: The capabilities of mass spectrometry for microscale determination of anthocyanins were investigated using high-performance liquid chromatography electrospray ionization mass spectrometry (LC-ESI/ MS) and tandem mass spectrometry (MS-MS). Four anthocyanins [cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3–(2^G−xylosylrutinoside) and cyanidin 3-rutinoside] were characterized in black raspberry samples by LC-ESI/MS-MS using both positive and negative ion analyses. Quantification of anthocyanins was conducted using ESI/MS-MS with selected reaction monitoring (SRM). Linear responses of several anthocyanins were determined during MS-MS analyses. Detection limits as low as 1 femtomol for most anthocyanins were obtained during ESI/MS-MS. Compared with other quantitative procedures such as high-performance liquid chromatography (HPLC) and ultraviolet/visible spectrophotometry, the current method provides an improved sensitive, specific technique for direct determination of intact anthocyanins. The developed methodology was successfully applied to analysis of trace levels of anthocyanins in human plasma and epithelial cells.     

Characterization of a new anthocyanin in black raspberries (Rubus occidentalis) by liquid chromatography electrospray ionization tandem mass spectrometry

Anthocyanin composition of black raspberry (Rubus occidentalis) was studied using high-performance liquid chromatography coupled to photodiode array (PDA) detection and electrospray ionization mass spectrometry (LC-ESI/MS) and tandem mass spectrometry (MS/MS). Pelargonidin 3-rutinoside was isolated and identified in black raspberries using HPLC, UV–Vis spectroscopy, MS, and NMR spectroscopy. No pelargonidin derivative had been previously found in Rubus occidentalis. In addition, the presence and identities of four previously reported anthocyanins (cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside) were confirmed by HPLC/MS and MS/MS analyses.     

红树莓果汁和桑椹果汁花色苷结构的鉴定及杀菌方式对果汁品质的影响

采用高效液相色谱-电喷雾质谱法和紫外-可见光谱法鉴定了红树莓及桑椹中主要花色苷及黄酮的组成,并 以红树莓果汁及桑椹果汁为原料,对比分析了经巴氏杀菌（pasteurization,PS）、煮沸杀菌（boiling sterilization, BS）、微波杀菌（microwave sterilization,MS）3 种杀菌方式处理前后其总花色苷、单个花色苷、总酚、主要黄 酮、H2O2的相对含量及其他理化性质（pH值、可溶性固形物含量、吸光度、褐变度、透光率）的变化。结果表 明：红树莓中的主要花色苷为矢车菊素-3-槐糖苷和矢车菊素-3-葡萄糖苷,桑椹中主要花色苷为矢车菊素-3-葡萄糖 苷和矢车菊素-3-芸香糖苷;与对照组相比,红树莓果汁和桑椹果汁总花色苷、单个花色苷及总酚的相对含量经3 种 杀菌方式处理后都有不同程度的降低,MS处理对其影响最小;3 种杀菌方式处理后的果汁中H2O2相对含量没有显 著性差别（P＞0.05）。MS处理可以较好地保持红树莓果汁和桑椹果汁的营养品质。     

Antioxidant capacities of individual and combined phenolics in a model system

In order to understand how interaction of individual phenolics contributes to the total antioxidant capacity, we quantitatively measured antioxidant capacity of various phenolics in different combinations, using ABTS radical-scavenging ability in a model system. Selected phenolics included in this study were those often found in fruits and vegetables, such as catechin, chlorogenic acid, cyanidin, cyanidin 3-glucoside, cyanidin 3-rutinoside, epicatechin, peonidin, peonidin 3-glucoside, quercetin, quercetin 3-glucoside, quercetin 3-galactoside and quercetin 3-rutinoside. Individual phenolics showed their characteristic antioxidant capacities, while the mixtures, with two or three phenolics combined revealed that the summation of antioxidant capacities of individual phenolics led to total antioxidant capacity. Therefore, it can be concluded that there was no synergistic effect among the phenolics studied. Only an additive effect of antioxidant capacity was observed.     

Investigations on Sweet Cherry Phenolic Degradation During Thermal Treatment Based on Fluorescence Spectroscopy and Inactivation Kinetics

Five anthocyanins were detected in the sweet cherry extract as follows: cyanidin 3-rutinoside, cyanidin 3-glucoside, peonidin 3-rutinoside, peonidin 3-glucoside, and pelargonidin 3-rutinoside, whereas the cyanidin 3-rutinoside was found to be in the highest amount. The effect of thermal treatment on the degradation of the polyphenolic compounds in sweet cherry extract was investigated in the range of 70–120 °C by means of fluorescence spectroscopy and spectrophotometric techniques. The fluorescence spectra were dominated by emission bands with maximum ranging from 356 nm at 25 °C to 350 nm at 110 °C. The heating of sweet cherry extract resulted in structural changes that led to a significant decrease in fluorescence intensity when increasing temperature. Degradation rate constants were estimated using a fractional conversion kinetic model. The activation energy values revealed a higher-temperature dependence of antioxidant activity, followed by anthocyanins, total polyphenols, and total flavonoids.     

Identification of anthocyanin pigments in strawberry (cv Camarosa) by LC using DAD and ESI-MS detection

New high performance liquid chromatography (HPLC) conditions were developed for the separation of strawberry anthocyanins that provided a good resolution of peaks at a low flow rate compatible with the requirements of the mass spectrometry (MS) detector. A strawberry extract was fractionated by column chromatography and simple fractions containing basically anthocyanins were obtained, making their analysis by HPLC possible using on-line photodiode array detection and MS. Information on the identity of the major and some secondary anthocyanins in strawberry was obtained from their retention characteristics, UV-visible spectra and mass spectra. The presence in strawberry of the previously reported cyanidin 3-glucoside, pelargonidin 3-glucoside, pelargonidin 3-rutinoside and pelargonidin 3-acetylglucoside was confirmed and cyanidin 3-rutinoside was identified in strawberry for the first time. Furthermore, cyanidin 3-malonyldiglucoside, pelargonidin 3-malylglucoside, a pelargonidin bioside and two possible pelargonidin 3-biosides acylated with acetic acid were also tentatively assigned.     

Fruit quality and bioactive compounds relevant to human health of sweet cherry (Prunus avium L.) cultivars grown in Italy

The fruit quality characteristics, phenolic compounds and antioxidant capacities of 24 sweet cherry (Prunus avium L.) cultivars grown on the mountainsides of the Etna volcano (Sicily, Italy) were evaluated. High-performance liquid chromatographic methods were used to identify and quantify sugars, organic acids and phenolics. A total of seven phenolic compounds were characterised as hydroxycinnamic acid derivatives (neochlorogenic acid, p-coumaroylquinic acid and chlorogenic acid) and anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, pelargonidin 3-rutinoside and peonidin 3-rutinoside). The total anthocyanin content ranged from 6.21 to 94.20 mg cyanidin 3-glucoside equivalents/100 g fresh weight (FW), while the total phenol content ranged from 84.96 to 162.21 mg gallic acid equivalents/100 g FW. The oxygen radical absorbance capacity (ORAC) assay indicated that fruit of all genotypes possessed considerable antioxidant activity. The high level of phenolic compounds and antioxidant capacity of some sweet cherry fruits implied that they might be sources of bioactive compounds that are relevant to human health.     

中压快速分离系统大量制备高纯度桑葚和树莓花色苷的工艺研究

为快速获得大量不同结构花色苷,本文以富含花色苷的桑葚和树莓为原料,通过大孔吸附树脂AB-8对两种花色苷粗提物初步分离后,利用中压快速分离系统分离得到高纯度的桑葚及树莓花色苷。制备条件为:以flash C₁₈(80 g,20~35 μm,100 A)为制备柱,两支串联,采用A相2%甲酸水,B相甲醇,流速30 mL/min,梯度洗脱程序:0~2 min,20% B;2~22 min,20%~30% B;22~32 min,30%~40% B,进样量300 mg,实现了3种不同结构花色苷的分离及纯化,桑葚中的矢车菊素-3-葡萄糖苷和矢车菊素-3-芸香糖苷产品纯度分别达到了95%和41%;树莓中的矢车菊素-3-槐糖苷和矢车菊素-3-葡萄糖苷产品纯度分别达到了60%和75%。其中桑葚中的矢车菊素-3-葡萄糖苷在32 min梯度程序内一次性可获得30 mg,且纯度为95%,可达到标准品的要求。     

红树莓花色苷含量测定及成分分析

运用HPLC-DAD-ESI-MS技术建立了测定红树莓花色苷含量的方法,并确定了花色苷的成分组成。采用Zorbax SB-C18色谱柱(250 mm×4.6 mm,5μm),甲醇-5%甲酸水溶液为流动相,流速为1.0 mL/min,检测波长为520 nm,以矢车菊素-3-葡萄糖苷为对照,用外标法定量,并通过紫外扫描光谱信息和ESI+碎片离子信息对花色苷组成成分定性分析。结果表明:红树莓总花色苷含量为105.69 mg/100 g,其主要含有的4种花色苷成分为矢车菊素-3-槐糖苷、矢车菊素-3-槐糖-5-鼠李糖苷、矢车菊素-3-葡萄糖苷、矢车菊素-3-芸香糖苷,相对含量分别为22.05%、13.83%、33.74%和30.38%。     

Involvement of Blueberry Peroxidase in the Mechanisms of Anthocyanin Degradation in Blueberry Juice

ABSTRACT: Addition of hydrogen peroxide (H₂ O₂) to blueberry juice changed the red coloration toward brown. Addition of ascorbic acid prevented the formation of brown polymers. An extract of peroxidase (POD) prepared from blueberry fruits was able to oxidize CG into the corresponding o-quinone but only in the presence of H₂ O₂. The chlorogenoquinone plays a dominant role in anthocyanin degradation. We demonstrated that peroxidase extract in the absence of CG showed a weak degradation activity toward blueberry anthocyanins, cyanidin 3-glucoside, and pelargonidin 3-glucoside. Nevertheless, addition of CG increased anthocyanin degradation, leading to formation of brown polymers. Therefore, blueberry POD could participate in the development of browning during blueberry-juice storage.     

Kinetics of anthocyanin degradation and polymeric colour formation in black carrot juice concentrates during storage

The changes in anthocyanins (ACNs) and polymeric colour of black carrot juice concentrate (BCJC) samples were monitored during storage at ?23, 5 and 20 °C for 319 days and at 30 °C for 53 days. While ACN degradation was fitted to a first‐order reaction model, polymeric colour formation was fitted to a zero‐order reaction model during the storage. Half‐life periods for ACN degradation in BCJCs were 603, 137 and 29 days at 5, 20 and 30 °C, respectively. The reaction rate constants for polymeric colour formation were 0.0207, 0.1435 and 0.5581%/days at 5, 20 and 30 °C, respectively. HPLC‐MS analyses of BCJC showed that cyanidin‐3‐galactoside‐xyloside‐glucoside‐ferulic acid (56%) was the major ACN, followed by cyanidin‐3‐galactoside‐xyloside (19%) and cyanidin‐3‐galactoside‐xyloside‐glucoside‐sinapic acid (10%). Cyanidin‐3‐galactoside‐xyloside‐glucoside‐ferulic acid was the most stable ACN in BCJC at storage temperatures. BCJCs should be kept at sub‐freezing temperatures to minimise ACN degradation.     

Antiproliferative effects of cherry juice and wine in Chinese hamster lung fibroblast cells and their phenolic constituents and antioxidant activities

Cherries are good sources of bioactive phenolic compounds that are widely considered to be potentially healthy. Here we investigated the protective activities of juice and wine products of tart and sweet cherries and their constituent anthocyanins (e.g., cyanidin 3-glucoside and cyanidin 3-rutinoside) against oxidative stress induced by hydrogen peroxide (H₂O₂) in Chinese hamster lung fibroblasts (V79-4). Total phenolics in the cherry juices and wines were 56.7–86.8 mg of gallic acid equivalents (GAE)/l and 79.4–149 mg GAE/l, respectively. Total anthocyanins in the cherry juices and wines were 7.9–50.1 mg of cyanidin 3-glucoside equivalents (CGE)/l and 29.6–63.4 mg CGE/l, respectively. Both cherry juices and wines exerted protective effects against oxidative stress induced by H₂O₂ on V79-4 cells and also enhanced the activities of antioxidative enzymes, such as superoxide dismutase and catalase, in a dose-dependent manner. The protection of V79-4 cells from oxidative stress by phenolics was mainly attributable to anthocyanins. The positive correlation between the protective effects against oxidative stress in V79-4 cells and the antioxidant enzyme activities was stronger for cyanidin 3-glucoside than for cyanidin 3-rutinoside.     

Effect on both aglycone and sugar moiety towards Phase II metabolism of anthocyanins

The effect of sugar moiety on anthocyanin metabolism was studied using anthocyanidin 3-rutinosides (cyanidin 3-O-rutinoside (Cy3R) and delphinidin 3-O-rutinoside (Dp3R)) and 3-O-glucosides (delphinidin 3-O-glucoside (Dp3G)). O-methylated Cy3R and Dp3R were detected in rat blood plasma after oral administration of Cy3R and Dp3R (100 mg/kg body weight). On the basis of HPLC retention time and UV–visible spectra together with the data of our previous studies on the hydrophobic metabolites of anthocyanidin 3-O-glucosides, it was concluded that both 3′- and 4′-O-methyl Cy3R were metabolites of Cy3R. On the other hand, only 4′-O-methyl Dp3R was detected as hydrophobic metabolite of Dp3R. A group of hydrophilic metabolites was also detected in rat blood plasma after oral administration of anthocyanins (Dp3G, Cy3R and Dp3R) and their structures were determined to be extended glucuronides and their O-methyl analogues by tandem MS analysis. The amounts of extended glucuronides of Dp3G, Cy3R and Dp3R were less than those of cyanidin 3-O-glucoside (Cy3G) reported in our previous study. On the other hand, anthocyanidin–glucuronides (both cyanidin–glucuronide and delphinidin–glucuronide) were not detected after oral administration of Cy3R, Dp3R and Dp3G. These results indicated that both the type of sugar moiety and stability of aglycone largely affected phase II metabolism of anthocyanins, and also indicated that the type of sugar moiety did not affect the O-methylation metabolism but affected glucuronyl conjugation in both liver and small intestine.     

Reaction Between Malvidin 3-Glucoside and (+)-Catechin in Model Solutions Containing Different Aldehydes

ABSTRACT: The contribution of the aldehyde composition of wine spirit to the color changes in Port red wine was studied in model solutions. Malvidin 3-glucoside was shown to be very reactive towards catechin in the presence of different aldehydes: acetaldehyde, isovaleraldehyde, benzaldehyde, propionaldehyde, isobutyraldehyde, formaldehyde, and 2-methylbutyraldehyde. LC/MS data confirmed the formation of oligomeric pigments resulting from the reaction between the anthocyanin and the flavanol (colored products) and between 2 flavanol units (colorless products) mediated by each aldehyde assayed. The UV-visible spectra of the colored pigments showed a λ_max bathochromically shifted relatively to the λ_max of original anthocyanins. All samples revealed a "blueing" and "darkening" color effects using the CIELAB system.