Optimization of microwave-assisted extraction of anthocyanins from purple corn (Zea mays L.) cob and identification with HPLC–MS

A Box–Behnken design was used to obtain the optimal conditions of microwave-assisted extraction (MAE). The effects of operating conditions, such as extraction time, solid–liquid ratio, and microwave irradiation power, on the extraction yield of anthocyanins were studied through a response surface methodology (RSM). The highest total anthocyainin content (TAC) from purple corn cob (185.1 mg/100 g) was obtained at an extraction time of 19 min, a solid–liquid ratio of 1:20, and a microwave irradiation power of 555 W. Six major kinds of anthocyanins were detected and identified as cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and their respective malonated counterparts. In comparison with the conventional solvent extraction, MAE was highly efficient and rapid in extracting anthocyanins from Chinese purple corn cob.Industrial relevanceIn the last decades, the interest in anthocyanin pigments has increased because of their possible utilization as natural food colorants and especially as antioxidant and anti-inflammatory agents. Purple corn cob was the byproduct during the corn processing. Purple corn cob is dark purple to almost black color due to its high content of anthocyanins, which makes this byproduct a good source of anthocyanins.     

黑玉米穗轴色素的分离及组成初探

采用纸层析、紫外可见光谱、红外光谱等技术对黑玉米穗轴色素进行分析研究,纸层析分离出三种主要成分,经纸层析、紫外可见光谱、红外光谱法初步确定分别是矢车菊-3-葡萄糖苷、芍药定-3-葡萄糖苷和天竺葵-3-葡萄糖苷。该黑玉米穗轴色素为花色苷类色素。     

Phenolic profiles of Andean purple corn (Zea mays L.)

Qualitative and quantitative high performance liquid chromatography with diode array detection (HPLC-DAD) was performed to characterize the presence of phenolic compounds in Andean purple corn. Phenolic compounds were analyzed by separating them in two main fractions: a water fraction (WF) and an ethyl acetate fraction (EAF). The WF rich in anthocyanins revealed the presence of cyanidin-3-glucoside, pelargonidin-3-glucoside and peonidin-3-glucoside. The respective acylated anthocyanin glucoside forms of these compounds were also detected following alkaline hydrolysis. The EAF was composed of phenolic acids such as p-coumaric, vanillic acid, protocatechuic acid, flavonoids such quercetin derivatives and a hesperitin derivative. Alkaline and acid hydrolysis of the EAF revealed the presence of p-coumaric and ferulic acid as main components in four bound hydroxycinnamic acid forms present in the ethyl acetate fraction.     

Effect of Ferulic Acid on the Formation of Pyranoanthocyanins from Purple Corn (Zea mays L.) Cob in a Model System and Their Effects on Color

The stability enhancing effect of ferulic acid on anthocyanins from purple corn cob was investigated in a model system. The addition of ferulic acid provided color-enhancing and stabilizing effects on purple corn cob during storage and heating. These effects were observed through changes in spectrophotometric and CIE L*a*b* color space parameters. In test samples enhanced with ferulic acid, high-performance liquid chromatography–mass spectrometry detected and identified three new anthocyanin-derived pigments, cyanidin-3-glucoside-vinylguaiacol, pelargonidin-3-glucoside-vinylguaiacol, and peonidin-3-glucoside-vinylguaiacol, with respective m/z values of 595, 579, and 609.     

Analytical characterization and the impact of ageing on anthocyanin composition and degradation in juices from five sour cherry cultivars

Five different sour cherry cultivars were processed to sour cherry juices in order to analyze their composition and the impact of storage on anthocyanin composition. Regarding primary juice parameters all cultivars showed high dry masses (13.8–17.2° Brix) and sugar free extracts (59.4–74.3 g/L). Anthocyanins were identified as cyanidin-3-(2^G-glucosylrutinoside), cyanidin-3-(2^G-xylosylrutinoside), cyanidin-3-sophoroside, cyanidin-3-glucoside, cyanidin-3-rutinoside, and peonidin-3-rutinoside. Besides the original anthocyanins, 5-carboxypyrano-anthocyanins occurred as new reaction products during storage. HPLC-MSⁿ experiments revealed, that a further ageing pigment was probably resulting from the reaction of an original anthocyanin with acetaldehyde. A significant decline of the original anthocyanin concentrations could be observed (70–75%) during 6 months of storage at 20 °C. Degradation kinetics of anthocyanins were calculated taking the reaction rate as first order. Colourless polyphenol concentrations and antioxidant capacities (TEAC) of the juices were not affected during storage.     

Identification of anthocyanins and distribution of flavonoids in tamarillo fruit (Cyphomandra betaceae (Cav.) Sendt.)

The major tamarillo (Cyphomandra betaceae) anthocyanin pigments were isolated and identified as pelargonidin-3-rutinoside, pelargonidin-3-glucoside, cyanidin-3-rutinoside, cyanidin-3-glucoside, delphinidin-3-rutinoside and delphinidin-3-glucoside. The intense purple-coloured jelly surrounding the seeds contained the greatest concentration of anthocyanins, delphinidin-3-rutinoside being the major pigment. Flavones, flavonols and leucoanthocyanins were also present in this material. The yellow-coloured flesh contained flavones and low concentration of anthocyanins. The major anthocyanin of the skins is cyanidin-3-rutinoside; flavones and leucoanthocyanins are also present. It is suggested that the presence of leucoanthocyanins in pigment extracts induced degradation of anthocyanins during isolation and purification.     

Phenolic Analyses of Haskap Berries (Lonicera caerulea L.): Spectrophotometry Versus High Performance Liquid Chromatography

Haskap berries (Lonicera caerulea L.) are known for their high phenolics, anthocyanins, and antioxidant potential. The data on the phenolic profile of these fruits are lacking. In this study, the phenolic profiles of three haskap varieties; tundra, berry blue, and indigo gem grown in Nova Scotia, Canada were investigated for the first time using spectrophotometery and high-performance liquid chromatography. Berries were analyzed for total phenolic content/total reducing capacity, total anthocyanin content, and antioxidant potential (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity). The total reducing capacity, total anthocyanin content, and 2,2-diphenyl-1-picrylhydrazyl values were 6.17–8.42 mg gallic acid equivalents/gram fresh weight, 4.49–6.97 mg cyanidin-3-glucoside equivalents/gram fresh weight, and 78.70–89.55%, respectively. The extracts were analyzed by reversed-phase diode array detector–high-performance liquid chromatography through a gradient elution using Synergi 4 µm Max-RP C12 column and the chromatograms were acquired at 520, 360, and 320 nm for athocyanins, flavonoids, and free phenolic acids, respectively. The identified anthocyanins were cyanidin-3-glucoside (82.81–91.99% of the total anthocyanins), cyanidin 3,5-di-glucoside (2.31–4.27%), cyanidin-3-rutinoside (1.54–9.20%), peonidin-3-O-glucoside (0.75–3.44%), and pelargonidin-3-glucoside (0.77–2.98%). Other flavonoids (quercetin-3-β-D-glucoside and quercetin-3-rutinoside) and free phenolic acids (chlorogenic and neochlorogenic) were also quantified.     

蔓越莓花色苷的组成鉴定及抗氧化能力

对蔓越莓花色苷的组成进行鉴定,并对其抗氧化能力进行测定。采用pH示差法测定花色苷提取量,超高压辅助提取蔓越莓花色苷含量为（75.49±0.43）mg/100?g,常规溶剂提取蔓越莓花色苷含量为（67.31±1.08）mg/100?g,蔓越莓中总花色苷含量为（79.52±0.50）mg/100?g;选择AB-8大孔树脂对蔓越莓花色苷粗提物进行纯化,冻干粉中花色苷含量从（46.10±0.92）mg/g提高到（309.26±2.37）mg/g。通过测定1,1-二苯基-2-三硝基苯肼自由基清除率、2,2’-联氮基-双-（3-乙基苯并噻唑啉-6-磺酸）二铵盐自由基清除能力和总抗氧化能力,比较蔓越莓花色苷与VC的抗氧化能力。结果表明：同质量浓度条件下,蔓越莓花色苷的抗氧化能力强于VC。通过高效液相色谱-质谱联用技术在蔓越莓中鉴定出7?种花色苷：芍药素-3,5-二己糖苷、矢车菊素-3-半乳糖苷、矢车菊素-3-葡萄糖苷、矢车菊素-3-阿拉伯糖苷、芍药素-3-半乳糖苷、芍药素-3-葡萄糖苷、芍药素-3-阿拉伯糖苷,其中芍药素-3,5-二己糖苷首次在蔓越莓中被鉴定出。     

基于液相色谱-质谱联用技术定性定量分析黑豆种皮中可溶型和结合型花青素

以超声波辅助有机溶剂提取法获得黑豆种皮可溶型花青素提取物,再进一步对不含可溶型花青素的黑豆种皮残渣使用酸水解和碱水解以及酸碱/碱酸轮提水解,获得黑豆种皮结合型花青素提取物。采用超高效液相色谱-电喷雾-四极杆飞行时间质谱联用仪分析鉴定各黑豆种皮提取物中所含有的共17 种花青素成分,包括11 种花青素糖苷类：飞燕草素-3-O-葡萄糖苷、飞燕草素-3-O-半乳糖苷、矮牵牛花素-3-O-葡萄糖苷、矮牵牛花素-3-O-半乳糖苷、天竺葵素-3-O-芸香糖苷、矢车菊素-3-O-葡萄糖苷、矢车菊素-3-O-半乳糖苷、天竺葵素-3-O-己糖苷、芍药花素-3-O-己糖苷、天竺葵素-3-O-(6”-丙二酰葡萄糖苷)和芹菜定-3-O-(6”-丙二酰葡萄糖苷);6 种花青素苷元：飞燕草素、矢车菊素、矮牵牛花素、天竺葵素、芹菜定和芍药花素。采用高效液相色谱-电喷雾-三重四极杆质谱联用仪精确定量各类黑豆种皮提取物中的花青素含量,结果表明,酸性结合型花青素提取物中结合型花青素的总含量最高。此外,在黑豆种皮的可溶型花青素提取物中,花青素主要以花青素糖苷类形式存在,苷元含量相对极少;而在结合型花青素提取物中,则主要以花青素苷元为主,糖苷类化合物相对少见。     

Short communication: Purple corn (Zea mays L.) stover silage with abundant anthocyanins transferring anthocyanin composition to the milk and increasing antioxidant status of lactating dairy goats

The present study used 16 multiparous lactating Saanen dairy goats (body weight, 41.80 ± 2.92 kg; mean ± standard deviation) with healthy and symmetrical udders. Goats were divided into 2 blocks of 8 goats based on milk yield averaged from 75 d in milk in a randomized completed block design. The 2 study groups were the control (CSSS), in which goats were fed sticky corn stover silage, and the treatment (TPSS), in which goats were fed anthocyanin-rich purple corn (Zea mays L.) stover silage (PSS). The results indicated that the TPSS group led to an elevation in the content of milk lactose relative to the CSSS. The inclusion of anthocyanin-rich PSS had no effect on the level of 5 particular anthocyanins [i.e., cyanidin-3-glucoside, delphinidin, cyanidin, pelargonidin (Pel), as well as total anthocyanins in milk]. The pelargonidin-3-glucoside and malvidin were unable to be detected in both groups. However, the TPSS resulted in higher levels of peonidin (Peo) and malvidin-3-O-glucoside (M3G) compared with the control. Moreover, goats receiving TPSS exhibited a higher level of superoxide dismutase (SOD) in plasma and milk relative to the CSSS. Interestingly, some positive correlations were detected between the certain milk components [i.e., fat and total solids as well as fat and solids-not-fat (SNF); protein and SNF; and total solids and SNF]. In addition, the positive correlations were observed between individual anthocyanins (cyanidin-3-glucoside, delphinidin, Peo, M3G, cyanidin, and Pel) and total anthocyanins. Specifically, stronger positive correlations were noted between several antioxidant enzymes and anthocyanin composition in milk (total antioxidant capacity and Pel; SOD and Peo as well as SOD and M3G). Taken together, PSS with abundant anthocyanins can transfer anthocyanins to the milk and enhance the amount of antioxidants in lactating dairy goats.     

Characterisation and preliminary bioactivity determination of Berberis boliviana Lechler fruit anthocyanins

Berberis boliviana Lechler is a member of the Berberidaceae family that has a small edible red-purple berry. The plant is native to the Peruvian Andes and contains high amounts of anthocyanin pigments. The monomeric anthocyanin content, determined by a pH-differential method, was 7/100 g of seedless berries. Pigments were characterised by HPLC coupled to a photodiode array (PDA) and mass spectrophotometer (MS) detectors. Five aglycones and ten anthocyanins were found and identified as petunidin-3-glucoside (24.4%), delphinidin-3-glucoside (24.1%), malvidin-3-glucoside (22.1%), cyanidin-3-glucoside (10.2%), petunidin-3-rutinoside (7.15%), malvidin-3-rutinoside (4.9%), cyanidin-3-rutinoside (3.8%), delphinidin-3-rutinoside (2.6%), peonidin-3-glucoside (1.1%), and peonidin-3-rutinoside (0.9%).     

蓝靛果酒发酵工艺优化及发酵过程对花色苷的影响

以蓝靛果为原料,进行蓝靛果酒发酵工艺的优化,并分析发酵过程对花色苷含量及花色苷组成的影响。通过比较不同酵母及不同糖类对果酒总酸、残糖、花色苷含量、乙醇体积分数及感官评分的影响,选择安琪葡萄酒果酒专用酵母SY作为发酵菌,蔗糖作为菌株的碳源,研究酵母接种量、起始pH值和发酵温度对蓝靛果酒理化性质及感官的影响,并在单因素试验的基础上进行3因素3水平的正交试验优化。结果表明,蓝靛果酒发酵的最佳工艺为：接种量0.15%、起始pH?3.2、发酵温度26?℃。在此条件下发酵12?d,乙醇体积分数为9.33%,感官评分为75.15,花色苷质量浓度为80.49?mg/L,为初始花色苷质量浓度（211.0?mg/L）的38.13%。利用高效液相色谱-串联质谱联用测定发酵对花色苷组成及各组成所占比例的影响,结果显示发酵前后的样品中均含有所测的8?种花色苷,发酵后矢车菊素-3-二己糖苷、芍药素-3,5-二己糖苷、矢车菊素-3,5-二己糖苷、芍药素-3-芸香苷、矢车菊素-3-乙酰基乙糖苷及芍药素-3-葡萄糖苷所占峰面积均有所增加,而矢车菊素-3-葡萄糖苷和天竺葵素-3-葡萄糖苷所占峰面积降低。     

Anthocyanin fingerprinting of true bokbunja (Rubus coreanus Miq.) fruit

The interest in black raspberry products has been increasing due to its flavor and potential health benefits. While black raspberries grown in North America are Rubus occidentalis, there has been some confusion regarding the identity of black raspberry grown in Korea (known as bokbunja; Rubus coreanus). As such, there is a need to define the anthocyanin profile of R. coreanus fruit collected from a verified source. We analyzed three genotypes of bokbunja fruit for anthocyanin profiles. While each varied in its anthocyanin proportions and total concentration, bokbunja fruit contained three anthocyanins: cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-glucoside, and lacked the xylose containing glycosides characteristic of R. occidentalis: cyanidin-3-sambubioside, and cyanidin-3-xylosylrutinoside. Due to mix-ups of bokbunja identity, research claiming to be conducted on bokbunja fruit requires confirmation that the fruit was sourced from a correctly identified plant. The distinct anthocyanin profiles between the two species can be used to confirm plant identity.     

‘蓓蕾’蓝靛果中花色苷组成鉴定及抗氧化能力比较分析

对蓝靛果中花色苷的组成进行鉴定,并对其抗氧化能力进行比较分析。实验以蓝靛果（‘蓓蕾’品种）为原料,采用有机溶剂60%乙醇（0.1%盐酸酸化）溶液,超声辅助提取90 min;利用D101大孔树脂对获得的粗提物进行纯化,之后冷冻干燥制得粉末物质。通过pH示差法和福林-酚法分别测定总花色苷含量和总多酚含量,分别为（353.35±0.79）、（474.01±2.12）mg/g;并用高效液相色谱-质谱联用法对花色苷组成进行鉴定,共发现11 种花色苷,其中矢车菊-3-葡萄糖苷为主要花色苷（90.679%）。此外,实验还通过总抗氧化能力测定和2,2’-联氨-二（3-乙基苯并噻唑-6-磺酸）二铵盐自由基、1,1-二苯基-2-三硝基苯肼自由基清除能力测定,比较分析蓝靛果花色苷提取物、矢车菊-3-葡萄糖苷、VC的抗氧化能力,结果表明,3 种物质的抗氧化能力排序为：矢车菊-3-葡萄糖苷＞花色苷提取物＞VC。     

Electrochemical behaviour and antioxidant capacity of anthocyanins from Chilean red wine,grape and raspberry

The anthocyanin fractions were extracted from Cabernet Sauvignon red wine, skins of Vitis vinifera grapes and raspberry fruits (Rubus idaeus). In red wine extract, 16 anthocyanins were identified, malvidin-3-O-glucoside being the main anthocyanin, which comprised 53.6% of the total anthocyanin in grape extract. Raspberry extract contained mainly delphinidin-3-O-glucoside and cyanidin-3-O-glucoside. The antioxidant capacity of the extracts was assayed by electrochemical methods. Best resolution of the oxidation peaks for the extracts and diluted wine was obtained by pulse differential voltammetry. The wine diluted 20× presented values of P1 (443 mV) and P2 (676 mV) similar to those corresponding to wine extract, and to the anthocyanin malvidin-3-O-glucoside. The antioxidant capacity of anthocyanins in extracts of wine, grape skin and raspberry fruit was also determined by the Trolox equivalent antioxidant capacity (TEAC) method.     

天然黑玉米色素研究与应用进展

黑玉米色素是从黑色玉米植株、玉米芯和玉米籽粒中提取花青苷,是一种安全、无毒天然食用色素;黑玉米色素呈有良好抗氧化活性,并具抗肿瘤、延缓衰老、降血压、调节血脂、降血糖等功能。黑玉米色素以酸性溶剂提取,其至少含有9种花色苷,主要活性物质为花青素-3-葡萄糖苷;黑玉米色素作为天然食品添加剂,可用于食品、药品、化妆品、染色剂等。该文综述黑玉米色素化学结构、理化性质、提取工艺及应用价值,对加强该色素深入研究和开发利用具有一定参考价值。     

Antioxidant assessment of an anthocyanin-enriched blackberry extract

Gel filtration of black berry (Rubus fruticosus sp) ethanolic extracts was employed to obtain an anthocyanin-enriched extract. The anthocyanin profile identified cyanidin-3-glucoside as the primary (e.g., 90% of total) anthocyanin present in blackberry. Gel filtration of crude extracts resulted in a 20-fold increase in total anthocyanin content, with no change in the proportion of cyanidin-3-glucoside. Antioxidant activities of both the crude and anthocyanin-enriched blackberry extracts were determined using cell-free (ORAC) and cell-based (INT-407 intracellular) antioxidant assays. Antioxidant activity, assessed by the ORAC assay, indicated a 7-fold increase in activity for the anthocyanin-enriched fraction. Similar results were obtained for the anthocyanin-enriched extract using the intracellular antioxidant assay with INT-407 cells. Our results indicate that the anthocyanin content, and more specifically the presence of cyanidin-3-glucoside, in blackberry, contributes a major part of the antioxidant ability to suppress both peroxyl radical-induced chemical and intracellular oxidation.     

Identification of anthocyanins isolated from black rice (Oryza sativa L.) and their degradation kinetics

Black rice is rich in anthocyanins-plant pigments. The aim of this work was to identify anthocyanins in black rice using high-performance liquid chromatography (HPLC)-electrospray ionization — mass spectrometry with diode array detection. Four different anthocyanins (cyanidin-3-glucoside, peonidin-3-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-rutinoside) were identified in black rice. Thermal stability of the four anthocyanins in black rice extract was studied at selected temperatures (80 °C, 90 °C and 100 °C) in the range of pH 1.0–pH 6.0. The results indicated that the thermal degradation of anthocyanins followed the first-order reaction kinetics. The temperature-dependent degradation was adequately modeled on the Arrhenius equation. The calculated values of activation energies (Ea), t_1/2 and k were different for the four anthocyanins. The degradation rate of monomeric anthocyanin increased with increasing heating temperature and pH values. Especially, as heating temperature increasing to 100 °C and pH value to 5.0.     

Characterisation of anthocyanins in the black soybean (Glycine max L.) by HPLC-DAD-ESI/MS analysis

The aim of this study was to isolate and identify the anthocyanins in the black seed coated soybean (cv. Cheongja 3, Glycine max (L.) Merr.) using reverse phase C-18 open column chromatography and high-performance liquid chromatography (HPLC) with diode array detection and electro spray ionization/mass spectrometry (DAD-ESI/MS) analysis, respectively. Anthocyanins were extracted from the coat of black soybeans with 1% TFA in methanol, isolated by RP-C-18 column chromatography, and their structures elucidated by 1D and 2D NMR spectroscopy. The isolated anthocyanins were characterised as delphinidin-3-O-glucoside (3), cyanidin-3-O-glucoside (5), petunidin-3-O-glucoside (6), pelargonidin-3-O-glucoside (7) and cyanidin (9). Furthermore, four minor anthocyanins were detected and identified as catechin-cyanidin-3-O-glucoside (1), delphinidin-3-O-galactoside (2), cyanidin-3-O-galactoside (4), and peonidin-3-O-glucoside (8) based on the fragmentation patterns of HPLC-DAD-ESI/MS analysis.     

Comparison of phenolic composition and antioxidant properties of two native Chilean and one domestic strawberry genotypes

The phenolic composition of extracts from the forms chiloensis and patagonica of Fragaria chiloensis were compared with that of the commercial strawberry Fragaria × ananassa cv. Chandler by high performance liquid chromatography with diode-array detector (HPLC–DAD) and high performance liquid chromatography with electrospray ionization mass spectroscopy detector (HPLC–ESI–MS). The phenolic constituents in the three species were mainly proanthocyanidins, hydrolysable tannins, anthocyanins and flavonol glycosides. In both native strawberry species the main flavonol glycoside was quercetin 3-O-glucuronide and the minor anthocyanins identified were cyanidin-malonyl-glucoside and pelargonidin-malonyl-glucoside. The highest anthocyanin content was found in the commercial red strawberry while ellagic acid was the main phenolic in the native white strawberry. From the methanolic fruit extract of the native Chilean strawberry Fragaria chiloensis ssp. chiloensis f. chiloensis four known antioxidants were isolated by selective fractionation using the bleaching of the free radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) as the guiding assay. The antioxidant properties (measured as the bleaching of the DPPH radical) were determined for methanol extracts of the three fruits, for fractions of F. chiloensis ssp. chiloensis f. chiloensis, and for the isolated compounds cyanidin-3-glucoside, pelargonidin-3-glucoside, quercetin-3-glucuronide and ellagic acid. This study allows a clear chemical differentiation between the commercial strawberry and the Chilean white strawberry.