Identification of anthocyanins in the sprouts of buckwheat

The anthocyanin profiles and varieties/breeding line differences of anthocyanin concentrations in common/tartary buckwheat sprouts have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-galactoside, and cyanidin 3-O-galactopyranosyl-rhamnoside, were isolated from the sprouts of common buckwheat, were separated using high-performance liquid chromatography (HPLC), and were identified using reversed-phase liquid chromatography (LC)/electrospray ionization-mass spectrometry (ESI-MS)/MS techniques. In tartary buckwheat sprouts, two anthocyanins (cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside) were identified. Among 19 common/tartary buckwheat varieties/breeding lines, Hokkai T10 contained the highest amounts of anthocyanins. Cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside concentrations in 6-10 days after seeding sprouts of Hokkai T10 ranged from 0.16 to 0.20 mg/g dry wt and from 5.55 to 6.57 mg/g dry wt, respectively. In addition, dark-grown sprouts of Hokkai T10 accumulated 0.091 and 2.77 mg/g dry wt of cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside whereas other varieties/breeding lines accumulated trace amounts of anthocyanins. Given its anthocyanin-rich red cotyledons, Hokkai T10 is a promising line for use as "Moyashi" type sprouts and is strongly recommended as a new functional food, rich in dietary anthocyanins.     

苦荞芽期黄酮合成关键酶和MYB转录因子基因的表达分析

苦荞(Fagopyrum tataricum)作为一种药食两用植物,富含以芦丁为主的黄酮类化合物.苦荞芽期芦丁含量较高,其分子机制尚不清楚.本研究选用西荞2号,采用AlCl3法测定了苦荞芽期6～10 d胚轴和子叶中的总黄酮,采用半定量RT-PCR分析其黄酮合成途径中主要关键酶基因苯丙氨酸氨裂解酶基因(Pal)、查尔酮异构酶基因(Chi)和黄酮醇合酶基因(Fls),以及MYB转录因子基因FtMyb1、FtMyb2和FtMyb3的相对表达水平,并对三者之间的相关性进行了统计学分析.结果表明,以相关系数绝对值大于0.75为阈值,子叶中,总黄酮的积累与FtMyb3表达显著正相关(0.9625),与FtMyb2表达显著负相关(-0.8572); Chi与FtMyb2表达显著正相关(0.8468),与FtMyb3表达显著负相关(-0.8010):Pal、Chi和Fls表达彼此显著正相关,相关系数分别为0.9119、0.8920和0.7584.子叶中总黄酮含量在4.58％～5.54％之间,且随芽期递增.Pal、Chi和Fls整体表达趋势相似,均呈现先升高后降低的趋势,且Fls的表达水平明显高于前二者.FtMyb2和FtMyb3整体表达趋势相反,FtMyb2呈下降趋势,FtMyb3呈上升趋势.胚轴中,总黄酮含量与Chi显著负相关(-0.8989); Fls与Chi显著负相关(-0.7498).结果提示,苦荞芽期黄酮合成的分子机制较为复杂,但部分基因表达仍存在显著相关性联系,为进一步选择苦荞分子操作靶位点提供参考.     

Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin

Two samples of tartary buckwheat (Fagopyrum tataricum Gaertn.) from China and one from Luxembourg were studied by high-performance liquid chromatography (HPLC) to reveal the possibilities of growing tartary buckwheat herb as a possible source of rutin, quercetin, and quercitrin. The content of rutin was determined as up to 3% dry weight (DW) in tartary buckwheat herb. Quercitrin values were in the range of 0.01-0.05% DW. Only traces of quercetin were detected in just some of the samples. Tartary buckwheat seeds contained more rutin (about 0.8-1.7% DW) than common buckwheat seeds (0.01% DW). Rutin and quercetin content in seeds depends on variety and growing conditions. Tartary buckwheat seeds contained traces of quercitrin and quercetin, which were not found in common buckwheat seeds.     

黑苦荞米黄酮提取工艺优化及其降血糖活性研究

为了获得合适的黑苦荞米黄酮提取工艺,该文采用单因素试验和响应面设计,选择乙醇体积分数、料液比、提取时间、提取温度4个因素,优化黑苦荞米黄酮提取工艺。试验结果表明,黑苦荞米黄酮的最佳提取条件是:乙醇体积分数54%,料液比1:24 g/m L,提取时间62 min,提取温度71℃。在此条件下,理论黄酮得率为2.21%,实际黄酮得率为2.20%,相对误差为0.45%。在此基础上进一步研究了黑苦荞米黄酮的α-淀粉酶抑制活性,结果表明7.5 mg/m L的黑苦荞米黄酮对α-淀粉酶活性的抑制率为54.05%,与二甲双胍(5 mg/m L)效果相当;此外,与空白对照相比,50μg/m L黑苦荞米黄酮能显著(P0.05)提高肝脏细胞Hep G2的葡萄糖消耗量(48.73%),并促进肝脏细胞糖原的合成。研究结果表明,黑苦荞米黄酮具有较好的辅助降血糖功效。     

棕色棉类黄酮3'-羟化酶基因(F3'H)的克隆及色素合成途径中相关基因表达特性研究

为探讨花色苷途径在彩棉色素形成中的作用及彩棉色素形成规律,本研究根据葡萄(Vitis vinifera)的类黄酮3'-羟化酶(flavonoid3'-hydroxylase,F3'H)基因全长cDNA序列blast所得棉花(Gossypium hirsutum)的EST序列(GenBank登录号:DT545210,CO071403和BG447485)设计引物,以开花后16d的新彩棉5号(XC-5)纤维为材料,采用RACE(rapid amplification of cDNA ends)和RT-PCR方法分离得到了2个类黄酮3'-羟化酶基因的全长cDNA序列,长度为1761和1892bp,均含有一个97~1629bp、长度为1533bp的开放阅读框,编码510个氨基酸,将这两个序列命名为GhF3'H-1和GhF3'H-2,分别提交GenBank,登录号为HM598123和HM598124,此2个序列编码区完全相同,仅在3'非翻译区(UTR)存在片段长短的差异。半定量RT-PCR检测花色苷合成途径中查尔酮合成酶(chalcone synathase,CHS)基因、F3'H、类黄酮3',5'-羟化酶(flavon...     

Cloning, characterization, and activity analysis of a flavonol synthase gene FtFLS1 and its association with flavonoid content in tartary buckwheat

Evidence from in vitro and in vivo studies indicates that rutin, the main flavonoid in tartary buckwheat ( Fagopyrum tataricum ), may have high value for medicine and health. This paper reports the finding of a flavonol synthase (FLS) gene, cloned and characterized from F. tataricum and designated FtFLS1, that is involved in rutin biosynthesis. The FtFLS1 gene was expressed in Escherichia coli BL21(DE3), and the recombinant soluble FtFLS1 protein had a relative molecular mass of 40 kDa. The purified recombinant protein showed, with dihydroquercetin as substrate, total and specific activities of 36.55 × 10(-3) IU and 18.94 × 10(-3) IU/mg, respectively, whereas the total and specific activities were 10.19 × 10(-3) IU and 5.28 × 10(-3) IU/mg, respectively, with dihydrokaempferol. RT-PCR revealed that during F. tataricum florescence there was an organ-specific expression pattern by the FtFLS1 gene, with similar trends in flavonoid content. These observations suggest that FtFLS1 in F. tataricum encodes a functional protein, which might play a key role in rutin biosynthesis.     

Antioxidant activity of tartary (Fagopyrum tataricum (L.) Gaertn.) and common (Fagopyrum esculentum moench) buckwheat sprouts

This study compared the differences of two types of buckwheat sprouts, namely, common buckwheat ( Fagopyrum esculentum Moench) and tartary buckwheat ( Fagopyrum tataricum (L.) Gaertn.), in general composition, functional components, and antioxidant capacity. The ethanol extracts of tartary buckwheat sprouts (TBS) had higher reducing power, free radical scavenging activity, and superoxide anion scavenging activity than those of common buckwheat sprouts (CBS). As for chelating effects on ferrous ions, CBS had higher values than TBS. Rutin was the major flavonoid found in these two types of buckwheat sprouts, and TBS was 5 fold higher in rutin than CBS. The antioxidant effects of buckwheat sprouts on human hepatoma HepG2 cells revealed that both of TBS and CBS could decrease the production of intracellular peroxide and remove the intracellular superoxide anions in HepG2 cells, but TBS reduced the cellular oxidative stress more effectively than CBS, possibly because of its higher rutin (and quercetin) content.     

水分胁迫下新造地施磷深度对苦荞生长及根系分布的影响

  【目的】  根系构型影响作物的抗旱能力,研究磷肥施用深度调节苦荞根系分布的可行性,为贫瘠干旱地区苦荞的生长提供科学养分管理措施。  【方法】  以‘黑丰1号’苦荞 (Fagopyrum tataricum L.) 为试验材料,进行根管土柱 (直径25 cm、高50 cm) 栽培试验,设置田间持水量65%～75% (W1)、45%～55% (W2) 和35%～45% (W3) 3种土壤水分条件,磷肥施用深度分别设置距离地表10 cm (P10)、20 cm (P20)、30 cm (P30) 以及3层均匀施用 (P-all) 4种方式,共有12个处理。在苦荞幼苗三叶一心期进行处理,生长22天后取样,测定根系构型,并记录生物量。  【结果】  干旱胁迫抑制了苦荞植株生长、干物质量的积累以及根系发育,其中W3水分条件抑制作用最为明显,导致苦荞株高、茎粗和叶面积较W1水分条件分别下降17.20%、18.03%和23.17%;根长、根表面积和根体积分别下降16.97%、20.39%和17.39%;地上部干物质量和根系干物质量分别下降39.16%、28.60%。干旱胁迫促进根系下扎,增加深层土壤中的根系分布。与W1水分条件相比,W2、W3水分条件下0—10和10—20 cm土层平均根长分别下降30.18%和27.55%、41.83%和41.02%,根系干物质量分别下降36.62%和33.61%、49.72%和48.11%;而20—30和30—45 cm土层中的苦荞平均根长则分别增加33.53%和42.52%、31.74%和50.95%,根系干物质量分别增加13.70%和26.84%、5.85%和28.64%。深层施磷促进施磷层土壤根系生长,与P-all处理相比,P10处理10—20 cm土层根长平均增加18.96%,P20处理20—30 cm土层平均增加32.39%,P30处理30—45 cm土层平均增加28.73%,根系干物质量依次分别增加26.62%、30.74%和24.65%。方差分析结果表明,各水分处理条件下,0—10和10—20 cm土层根系干物质量均表现为P10处理显著高于其他施磷处理,且其他处理间差异也达显著水平;而20—30、30—45 cm土层根系干物质量则表现为P20、P30施磷处理显著高于其他处理。  【结论】  水分和施磷深度对苦荞苗期植株生长以及根系分布均有显著影响。在干旱胁迫下,增加磷肥的施用深度可促进苦荞根系在20—45 cm深土壤中的分布,显著扩大根系对土壤养分和水分的获取空间,并最终促进苦荞的生长。本试验条件下,采样仅限于苦荞苗期,在水分胁迫条件下磷肥以10 cm的施肥深度效果最佳。     

Fagopyritols, D-chiro-inositol, and other soluble carbohydrates in buckwheat seed milling fractions

Fagopyritols are mono-, di-, and trigalactosyl derivatives of D-chiro-inositol that accumulate in seeds of common buckwheat (Fagopyrum esculentum Moench) and may be important for seed maturation and as a dietary supplement. Fagopyritols and other soluble carbohydrates were assayed in mature groats and 11 milling fractions of common buckwheat seed. Because fagopyritols are in embryo and aleurone tissues, differences in fagopyritol concentrations reflect varying proportions of these tissues in each milling fraction. Bran milling fractions contained 6.4 g of total soluble carbohydrates per 100 g of dry weight, 55% of which was sucrose and 40% fagopyritols. Flour milling fractions had reduced fagopyritol concentration [0.7 g/100 g of dry weight total fagopyritols in the dark (Supreme) flour and 0.3 g/100 g in the light (Fancy) flours]. Fagopyritol B1 was 70% of total fagopyritols in all milling fractions. Fagopyritols were 40% of total soluble carbohydrates in groats of two cultivars of common buckwheat but 21% in groats of tartary buckwheat [Fagopyrum tataricum (L.) Gaertn.], probably a reflection of environment and genetics. A rhamnoglucoside present in tartary buckwheat was not detected in common buckwheat.     

Pleiotropic effect of phenolic compounds content increases in transgenic flax plant

The principal goal of this paper was to generate flax (Linum usitatissimum L.) plants with increased antioxidant properties. To accomplish this a vector containing a multigene construct was prepared, and transgenic plants overexpressing essential flavonoid biosynthesis pathway enzymes were generated and analyzed. The simultaneous expression of genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) resulted in a significant increase of flax antioxidant capacity. To investigate the determinants of higher antioxidant properties of transgenic plants, the phenolic acids and lignans compound contents were measured. In both green part and seed extracts from transgenic plants, the phenolic acids level was increased when compared to the control. The calculated correlation coefficient between phenolic acids content and antioxidant capacity (0.82 and 0.70 for green part and flaxseed, respectively) perfectly reflects their strong relationship. The increase in yield of transgenic plants and their higher resistance to Fusarium culmorum and Fusarium oxysporum when compared to the control plants was a characteristic feature. It was assessed a very high correlation (correlation coefficient = 0.9) between phenolic acids level in flaxseed extract and resistance to F. culmorum. The flowering date of transgenic plants was approximately 3 weeks earlier than that of the control plants. Interestingly, a significant increase in monounsaturated fatty acids and a slight increase in lignans content accompanied the increase in antioxidant properties of flaxseeds.     

Influence of P Deficiency on Major Secondary Metabolism in Flavonoids Synthesis Pathway of Chrysanthemum morifolium Ramat

The aim of this paper is to study the influence of P deficiency on secondary metabolites of flavonoid in Chrysanthemum morifolium Ramat. The regression equation between flavonoid and phenolic acid content was Y = ?294.46X + 150.66 (R² = 0.9205, P < 0.01) in check and Y = 42.62X + 2.49 (R² = 0.9564, P < 0.01) in P deficient treatment (Y, flavonoid content; X, phenolic content). There were two principal components to control the flavonoid major synthesis process, that was principal 1 [phenolic acid, phenylalanine ammonia-lyase (PAL), cinnamic acid and p-coumaric acid] (88.17%) and principal 2 [4-coumarate coenzyme A ligase (4CL), Cinnamate-4-hydroxylase (C4H)] (9.64%) domination under normal growth of C. moriloium. However, under P deficiency condition, the principal components were principal 1 (phenolic acid, cinnamic acid and p-coumaric acid and C4H) (81.46%) and principal 2 (PAL) (18.53%) domination, respectively. The influence of P deficiency on flavonoid major synthesis pathway was caused by the change of PAL and C4H activities.     

Protective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone to PC12 cells against cytotoxicity induced by hydrogen peroxide

Oxidative stress has been considered as a major cause of cellular injuries in various clinical abnormalities. One of the possible ways to prevent reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical therapies to augment the endogenous antioxidant defense capacity. The present study found that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone isolated from the buds of Cleistocalyx operculatus, possessed cytoprotective activity in PC12 cells treated with H(2)O(2). The results showed that DMC could effectively increase cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction], decrease the cell apoptotic percentage [annexin V/propidium iodide (AV/PI) assay], prevent the membrane from damage [lactate dehydrogenase (LDH) release], scavenge ROS formation, reduce caspase-3 activity, and attenuate the decrease of mitochondrial membrane potential (MMP) in PC12 cells treated with H(2)O(2). Meanwhile, DMC increased the catalytic activity of superoxide dismutase (SOD) and the cellular amount of glutathione (GSH), decreased the cellular amount of malondialdehyde (MDA), and decreased the production of lipid peroxidation in PC12 cells treated with H(2)O(2).     

Color and antioxidant properties of cyanidin-based anthocyanin pigments

A series of cyanidin-based anthocyanin pigments was investigated to determine the effect of structural variation on a number of chemical and physical properties: CIELAB color coordinates, visual detection thresholds, hydration constants (pK(H)), and in vitro antioxidant activities (ORAC). In addition to individual isolated compounds, purified total pigment isolates from blackberry, elderberry, black carrot, red cabbage, and sweet potato were also examined. Acylation with cinnamic acids shifted color tonality (hue angle) to purple, and markedly increased pK(H) and antioxidant activity, but lowered the visual detection threshold. Glycosidic substitution at the 5 position moved tonalities toward purple and decreased pK(H), and tended to lower the ORAC value, but raised the visual detection threshold. Increasing the number of sugar substituents at the 3 position also affected all of these parameters, however, the extent was not predictable. Antioxidant levels of purified anthocyanin extracts were much higher than expected from anthocyanin content indicating synergistic effect of anthocyanin mixtures.     

Variation of major minerals and trace elements in seeds of tartary buckwheat (Fagopyrum tataricum Gaertn.)

Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains protein of high nutritional value, polyphenols, vitamins and minerals. It is one of the most important minor crops in China and has a great potential as a health and functional food. However, information on the elemental mineral composition of its seeds remains limited. The concentrations of Cu, Zn, Fe, K, and Mg in seeds of 123 tartary buckwheat accessions from the same cultivation were studied by means of flame atomic absorption spectrometry. The results revealed that the average concentrations of Cu (x₁), Zn (x₂), Fe (x₃), K (x₄), and Mg (x₅) elements in the accessions are 19.49 (with a range of 5.74–36.01 mg/kg), 27.41 (8.44–66.63 mg/kg), 656.24 (21.8–3,990 mg/kg), 3,639.23 (1,737–5,831 mg/kg), and 1,523.89 mg/kg (729–3,104 mg/kg) respectively. Among them, Fe concentration has the highest coefficient of variation (114.7 %). The results also revealed five significant positive correlations among Cu, Zn, Fe, K, and Mg concentrations. Therefore, distinct genotypes with high concentration of mineral elements should be effective for the development of special buckwheat varieties and improvement of its food nutritional quality.     

Expression, cloning, and immunological analysis of buckwheat (Fagopyrum esculentum Moench) seed storage proteins

cDNA of buckwheat (Fagopyrum esculentum Moench) was isolated from immature seeds harvested 14 days after pollination. Two genes, designated FA02 and FA18, were found to encode legumin-like proteins and were expressed during seed development. The deduced amino acid sequence of FA02 was identical to the N-terminal amino acid domain of BW24KD, which was believed to be a major buckwheat allergen (Urisu, A.; Kondo, Y.; Morita, Y.; Yagi, E.; Tsuruta, M.; Yasaki, T.; Yamada, K.; Kuzuya, H.; Suzuki, M.; Titani, K.; Kurosawa, K. Isolation and characterization of a major allergen in buckwheat seeds. In Current Advances in Buckwheat Research; Shinshu University Press: Matsumoto, Japan, 1995; pp 965--974). It was predicted that FA02 would be cleaved to generate two separate components, a 41.3 kDa alpha-subunit and a 21 kDa beta-subunit. Antiserum was raised against the deduced FA02 beta-subunit, and immunoblotting of total protein from buckwheat seeds (F. esculentum M. and Fagopyrum tartaricum Gaertn.) revealed that several groups of proteins reacted with the antiserum. Polypeptides in the 23--25 kDa range displayed the greatest reactivity.     

Antioxidant activity of black currant anthocyanin aglycons and their glycosides measured by chemiluminescence in a neutral pH region and in human plasma

The antioxidant activity of nine anthocyanin glycosides was measured in a neutral pH region using a chemiluminescence (CL) emission system in the presence of an H(2)O(2)-acetaldehyde system, and the intensities were found to be affected by three factors, pH value and both moieties of the aglycon and C-3 sugar. With an increase in pH from 4.0 to 9.0, the CL intensities increased from pH 5.0, reached their maxima at pH 6.0-7.0, and decreased at pH 9.0. Comparison of the intensities among the 3-glucosides with five different aglycons and the 3-glycosides with three different sugar moieties at C-3 showed that their strongest intensities were given by the delphinidin aglycon and 3-rutinosyl moiety, respectively. Monitoring of the CL intensity of human blood plasma for 8 h after oral administration of black currant anthocyanins (BCA) showed a rapid increase until 2 h, and a significant difference (P < 0.05) was recognized at 1-8 h.