桑葚花色苷的分离纯化及其热降解动力学研究

通过AB-8大孔树脂、乙酸乙酯萃取和Toyopearl TSK HW-40S凝胶柱层析对桑葚汁中的花色苷进行分离纯化,得到两个单一的化合物组分。经HPLC-MS鉴定这两种花色苷分别为矢车菊素-3-芸香糖苷和矢车菊素-3-葡萄糖苷。在此基础上,研究了不同纯度花色苷的降解动力学,结果表明:桑葚花色苷粗提物中所含的黄酮类化合物对花色苷的热降解有较强的保护作用,含黄酮的花色苷体系对p H值的变化更为敏感。在p H 3.5时,矢车菊素-3-芸香糖苷和矢车菊素-3-葡萄糖苷的热稳定性基本相同;在p H 4.0时,矢车菊素-3-芸香糖苷的热稳定性略好于矢车菊素-3-葡萄糖苷。     

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%。     

黑豆皮花色苷的中压液相色谱制备及结构鉴定

以黑豆皮为实验材料,用乙醇浸提法对黑豆皮中的花色苷进行提取,用大孔吸附树脂对花色苷进行纯化,经冷冻干燥得到黑豆皮花色苷粗品。利用中压制备色谱对花色苷组分进行分离,通过质谱分析鉴定经中压制备色谱分离后的花色苷组分。结果表明:黑豆皮花色苷粗品中的总花色苷含量为26.9%,经中压制备色谱对花色苷粗品进行分离后的2峰中矢车菊素-3-葡萄糖苷纯度达到91.46%。黑豆皮中的主要花色苷为天竺葵素-3-O-芸香糖苷、芍药色素-3-O-葡萄糖苷、矢车菊素-3-O-葡萄糖苷和锦葵素-3-葡萄糖苷-4-乙醛。     

野生桑葚中花色苷成分分析

运用固相萃取纯化技术与高效液相色谱/二极管阵列检测器/电喷雾质谱联用技术,采用ZorbaxSB-C18色谱柱(250mm×4.6mm,5μm),甲醇-5%甲酸水溶液为流动相,流速为1.0mL/min,检测波长为520nm,以矢车菊素3-葡萄糖苷为对照,外标法测定了野生桑葚花色苷含量,并通过紫外扫描光谱和电喷雾质谱正离子碎片信息确定了花色苷的成分组成。结果表明:野生桑葚总花色苷含量为154.27mg/100g,含有的三种花色苷成分分别为矢车菊素3-葡萄糖苷、矢车菊素3-芸香糖苷和天竺葵素3-葡萄糖苷,其相对含量为67.52%、31.29%和1.06%。     

不同花色苷代表性成分的分离鉴定及体外抗氧化活性

本实验以紫甘薯、黑枸杞、黑加仑和桑葚花色苷提取物为原料,制备其单体花色苷组分并研究其体外抗氧化性质。选取每种花色苷中含量较高,分子量居中,具有代表性的单体化合物作为目标组分,采用高速逆流色谱制备分离四种来源的花色苷。选用甲基叔丁基醚-正丁醇-乙腈-水-三氟乙酸作为溶剂体系,流速设定为5 mL/min,转速为850 r/min,分离得到高纯度花色苷单体化合物。采用分光光度法、HPLC-MS法分析测定花色苷含量及主要组成,用DPPH自由基、羟自由基清除能力和总还原力的测定分析其体外抗氧化活性。结果表明,四种来源的花色苷中代表性的成分依次为芍药素-3-咖啡酰-阿魏酰槐糖苷-5-葡萄糖苷、矮牵牛素-3-O-对香豆酰芸香糖苷-5-O-葡萄糖苷、矢车菊-3-芸香糖苷和矢车菊-3-O-葡萄糖苷,它们均具有良好的体外抗氧化活性。     

利用中压制备液相色谱从桑葚中快速制备矢车菊素-3-葡萄糖苷单体

单体花色苷的快速大量制备长久以来是花色苷产业化中的难题,而中压制备液相色谱在产业应用中有着很大的开发空间。选取花色苷组分较单一的桑葚为实验原料,经提取分离总花色苷后使用填装有反相C18填料的耐压玻璃柱作为中压制备液相色谱柱,纯化制备矢车菊素-3-葡萄糖苷单体。结果显示：3 个色谱分离峰中目的峰（峰2）经高效液相色谱和质谱确证为由矢车菊素-3-葡萄糖苷（cyanidin-3-glucoside,C3G）和矢车菊素-3-芸香糖苷（cyanidin-3-rutinoside,C3R）组成,采用峰面积归一化法计算得到C3G纯度为73.56%;通过对峰2采用切割方式进行收集,C3G纯度达到98%以上,单次收集到C3G单体溶液650?mL。中压制备液相色谱法单次上样量大、步骤简洁、成本低廉,可为矢车菊素-3-葡萄糖苷单体的规模化生产提供一定的参考。     

超声辅助果胶酶法提取红树莓花色苷及其成分测定

本研究利用超声辅助果胶酶法来提取制备红树莓花色苷,通过单因素实验,研究花色苷提取工艺中果胶酶浓度、料液比、酶解pH、酶解温度、超声时间和超声功率对提取液中花色苷含量的影响,结合响应面实验对提取工艺进行了优化,对比超声辅助果胶酶法和单一提取法所得的花色苷含量,并利用超高效液相色谱仪串联四级杆/飞行时间质谱(UPLC-Q/TOF)对树莓花色苷进行结构鉴定。结果表明:红树莓花色苷最佳提取条件为:果胶酶浓度5 mg/g、料液比1:15(g/mL)、酶解pH3、酶解温度50℃、酶解时间60 min、超声时间20 min和超声功率450 W,此时所得花色苷含量为127.51 mg/100 g。超声辅助酶法所得到花色苷含量较酶法提高了24.58 mg/100 g,较超声法提高40.40 mg/100 g,较单一提取法,超声辅助酶法具有更好的提取效果。经过超高效液相色谱三重四级杆飞行质谱分离中主要花色苷为:芍药素-3-葡萄糖苷、矢车菊素-3-葡萄糖苷、矢车菊素-3-阿拉伯糖苷和矢车菊素-3-芸香糖苷。     

采用HPLC-DAD-ESIMS技术鉴定紫山药中的花色苷成分

采用大孔树酯对浙江台州黄岩紫山药中的花色苷进行分离,并用高压液相色谱-二极管阵列检测-质谱检测技术对其中的主要花色苷进行结构鉴定。通过差异pH法分析紫山药中花色苷的含量为28.8mg/kg。紫山药中4种主要的花色苷可能是:矢车菊素-3-葡萄糖苷或半乳糖苷的双咖啡酸酰化物,矢车菊素-3-二糖苷的芥子酸酰化物,矢车菊素-3-二糖苷阿魏酸的酰化物,芍药色素-3-二糖苷的芥子酸酰化物。     

云南丽江和吉林靖宇11种越橘果实花色苷组分的分析

采用高效液相色谱法对云南丽江、吉林靖宇11种越橘果实花色苷组分进行测定。通过标准品的分析,建立了飞燕草素-3-半乳糖苷、飞燕草素-3-葡萄糖苷、矢车菊素-3-半乳糖苷、矢车菊素-3-葡萄糖苷、锦葵色素-3-半乳糖苷、锦葵色素-3-葡萄糖苷的回归方程,相关系数为0.9939~0.9968,精密度RSD为2.00%~3.73%,回收率在98.90%~100.99%,方法准确可行。通过6种标准品的对比,供试的15个样品中,所有越橘品种均有飞燕草素-3-半乳糖苷、矢车菊素-3-半乳糖苷、锦葵色素-3-半乳糖苷3种花色苷,飞燕草素-3-半乳糖苷平均含量为223.99 μg/g、锦葵色素-3-半乳糖苷平均含量为153.34 μg/g,二者占总花色苷的65%。采用欧氏距离聚类分析表明,集群1为飞燕草素-3-半乳糖苷含量较高的三种越橘,代表品种为丽江雷戈西、丽江奥尼尔、靖宇杜克;集群2为6种花色苷总含量较高的越橘品种,代表品种为丽江北陆与靖宇早蓝,说明飞燕草素-3-半乳糖苷含量与花色苷总含量是评价越橘花色苷特点的重要因子。     

UPLC-Q-Tof/MS同时测定桑葚保健酒中五种花色苷含量

目的:建立超高效液相色谱-四级杆-飞行时间质谱(UPLC-Q-Tof/MS)法测定桑葚保健酒中五种花色苷(天竺葵-3-O葡萄糖苷、氯化芍药素-3-O-葡萄糖苷、矢车菊-3-O芸香葡萄糖苷、锦葵色素-3-5-二葡萄糖苷、矢车菊-3-O葡萄糖)含量。方法:色谱柱:ACQUITY UPLC Ben C18 Column (2.1mm×100mm,1.7μm),流动相为乙腈-0.1%甲酸,进样量为2μL,流速为0.5mL/min,梯度洗脱,柱温为40℃,ESI+电离模式。结果:五种花色苷在100～800ng/mL范围内线性关系、回收率良好。桑葚保健酒中矢车菊-3-O-芸香葡萄糖苷、矢车菊-3-O-葡萄糖苷含量丰富,达到398.0ng/mL以及532.8ng/mL。     

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。     

Influence of alcoholic fermentation process on antioxidant activity and phenolic levels from mulberries (Morus nigra L.)

The aim of the present study was to evaluate the profile of the phenolic constituents of Morus nigra fruits and their antioxidant activity (DPPH) and to compare their contents before and after fermentation. Antioxidant phenolics of black mulberry (M. nigra L.) samples grown in Galicia (NW Spain) were extracted with methanol/formic acid/water (MFW) and determined by high performance liquid chromatography (HPLC). Two major anthocyanins (cyanidin 3-glucoside and cyanidin 3-rutinoside) and two flavonols (quercetin 3-glucoside and rutin) were isolated, together with caffeic acid and other hydroxycinnamic and ellagic acid derivatives. Their chemical structures were identified by spectral analyses with diode array detection (DAD), but also with alkaline saponification and acid hydrolysis of the mulberry phenolics. Good correlations (r² = 0.6229) were observed among total flavonols contents and the IC₅₀ radical scavenging capacities of mulberry fruits. Anthocyanins are the major flavonoids present in mulberry. It would be expected that anthocyanins contribute significantly to their antioxidant activity; nevertheless, alcohol generated during fermentation may also contribute to antioxidant activity. Our results provide useful antioxidant nutritional information of fresh and fermented mulberry fruits.     

Anthocyanins and Other Phenolic Compounds of Saskatoon Berries Amelanchier alnifolia Nutt.)

High performance liquid chromatography (HPLC), equipped with photodiode array detector, was applied to separation and concentration of the anthocyanins and related compounds of saskatoon berries. Thirteen discrete peaks were detected and separation of these took less than 40 min. Ten of the 13 HPLC peaks were collected as separate fractions from the column outlet and re-analyzed by paper chromatography and spectral analysis. The concentration of total anthocyanins was 86-125 mg/100g of fresh berries. The main anthocyanins were cyanidin 3-galactoside, which formed 61% and cyanidin 3-glucoside which accounted for 21% of the total anthocyanins. Also identified were cyanindin 3-xyloside, chlorogenic acid and rutin.     

Optimization of microwave-assisted extraction of anthocyanins from mulberry and identification of anthocyanins in extract using HPLC-ESI-MS

Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants. This study aims to optimize the microwave-assisted extraction (MAE) conditions of anthocyanins from mulberry (M. atropurpurea Roxb.) using response surface methodology (RSM). A Box-Behnken experiment was employed in this regard. Methanol concentration, microwave power, and extraction time were chosen as independent variables. The optimized conditions of MAE were as follows: 59.6% acidified methanol, 425 W power, 25 (v/w) liquid-to-solid ratio, and 132 s time. Under these conditions, 54.72 mg anthocyanins were obtained from 1.0 g mulberry powder. Furthermore, 8 anthocyanins were identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) in mulberry extract. The results showed that cyanidin-3-glucoside and cyanidin-3-rutinoside are the major anthocyanins in mulberry. In addition, in comparison with conventional extraction, MAE is more rapid and efficient for extracting anthocyanins from mulberry.     

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.     

Purification of Anthocyanins from Extracts of Red Raspberry Using Macroporous Resin

The performances and separation characteristics of nine widely used macroporous resins for the enrichment and purification of anthocyanins from red raspberries extracts were investigated. AB-8 resin offered the maximum adsorption and desorption behavior for anthocyanins among the resins tested, and its adsorption behavior was fitted to the Langmuir and Freundlich isotherms at different temperatures. In order to optimize the operating process, the dynamic adsorption and desorption experiments were carried out on an AB-8 resin-packed column. The optimum parameters for adsorption were sample solution anthocyanins concentration 0.2912 mg/mL, processing volume 3.5 BV, flow rate 0.5 mL/min; optimum parameters for desorption were elution solvent ethanol–water (60:40, v/v) 2.5 BV and flow rate 1.0 mL/min. After one run treatment with AB-8 resin, the anthocyanins purity increased 19.1-fold with a recovery yield of 98.84%. Two kinds of anthocyanins were obtained by further processing with a Sephadex LH-20 column, which were identified as cyanidin-3-glucoside and cyanidin-3-sophoroside using high-performance liquid chromatography–mass spectrometry, and the purity was 95.52 and 94.76%, respectively. The separation process developed via column chromatography in this study provided a potential approach for scale-up purification of anthocyanins from red raspberries or other similar berries.     

Rapid determination of free anthocyanins in foodstuffs using high performance liquid chromatography

Anthocyanins constitute a major group of natural pigments, and they are responsible for the colours of fruits and vegetables. A rapid and feasible assay procedure for the determination of free forms of the six most abundant anthocyanins in foods is described. The 3-glucoside forms of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin with the aglycone cyanidin (as internal standard) were separated by gradient elution and quantified using HPLC-DAD within 18 min. A fast sample preparation step was employed which allows direct injection of samples to the chromatograph without need of chemical extraction. Testing on 28 different vegetable, fruit and processed commercial product samples demonstrated applicability in the concentration range of about 80–420 ng/mL with an accuracy of 99.2 ± 0.2% and an average precision of 0.8%. The method was suggested as a cheap and robust alternative to the previous ones that employ multi-step sample treatment protocols.     

桑葚花色苷超高压提取工艺优化及其组分分析

以桑葚为原料采用超高压法提取花色苷,通过响应面优化提取工艺,并用高效液相色谱法分析桑葚提取液中花色苷种类和含量。结果表明:超高压提取桑葚花色苷的最佳工艺条件为:压力270 MPa、保压时间6 min、料液比1:35 g/mL、乙醇浓度62%,在此条件下桑葚花色苷得率为(4.93±0.12)mg/g,较热回流提取和超声波提取得率分别提高了25.93%和18.26%。从桑葚提取液中鉴定出4种花色苷单体分别为飞燕草色素、矢车菊色素、牵牛花色素、芍药色素,其含量分别为1.32、2.05、0.27、0.25 mg/g。高效液相色谱分析花色苷组分为桑葚花色苷进一步开发利用提供理论依据。     

Jam Processing Effect on Phenolics and Antioxidant Capacity in Anthocyanin-rich Fruits: Cherry, Plum, and Raspberry

ABSTRACT: Four cultivars of sour cherries (Balaton, Karneol, Kroeker and Northstar), 2 cultivars of plums (BY 8158.50 and Methley), and 1 red raspberry cv. Prelude were analyzed for total phenolics, antioxidant capacity, and total anthocyanins before and after jam production to evaluate their changes after thermal processing. Fruits had total phenolics ranging from 245.7 to 398.5 mg gallic acid equivalent (GAE)/100 g. Antioxidant capacity of fruits ranged from 354.8 to 692.3 mg/100 g, expressed as vitamin C equivalent antioxidant capacity (VCEAC). Total anthocyanins of fruits ranged from 30.9 to 67.1 mg cyanidin 3-glucoside equivalent (CGE)/100 g. In 100 g of jam, total phenolics varied from 132.9 to 218.9 mg GAE, while antioxidant capacity ranged from 205.6 to 373.5 mg/100 g VCEAC. Jams had total anthocyanins of 5.4 to 30.4 mg CGE/100 g. On the basis of fresh fruit (100 g), the processing and heating during jam making generally decreased the contents of total phenolics, VCEAC, and total anthocyanins. Major losses occurred in anthocyanin content where overall retention varied from 89% to 21%. HPLC analysis of individual anthocyanins from cherry cv. Balaton to its jam showed that processing caused 90% decrease in anthocyanins. The results indicated that more than 73% total phenolics and more than 65% antioxidant capacity were retained after processing fruits into jams. Optimization of food processing would help to conserve the bioactive phenolic compounds in fruits.