响应面优化超临界CO2萃取陈皮多甲氧基黄酮研究

选用江西产陈皮作为原料,用超临界CO_2流体对其中的多甲氧基黄酮进行萃取,并对最佳萃取工艺条件进行探讨。以川陈皮素、3,5,6,7,8,3',4'-七多甲氧基黄酮和橘皮素3种主要多甲氧基黄酮总量作为陈皮中总黄酮含量指标,通过单因素试验和响应面试验设计优化萃取工艺,确定陈皮中多甲氧基黄酮最佳萃取工艺为:压力28 MPa、温度55℃、时间67 min。在此优化条件下,从陈皮中萃取3种主要多甲氧基黄酮总得率为1.89 mg/g。     

陈皮中川陈皮素的提取分离及改性

以陈皮中川陈皮素的提取、分离纯化以及改性为目的,采用无水乙醇浸提陈皮得浸膏,以氯仿为萃取剂萃取浸膏,得到多甲氧基黄酮混合物,再以硅胶为吸附剂、乙酸乙酯和石油醚的混合液为洗脱剂,采用柱色谱法分离多甲氧基黄酮化合物,经高效液相色谱和气相色谱-质谱检测并与标准品对比,最终确证得到川陈皮素纯品。研究从川陈皮素到5-去甲基川陈皮素的合成路线,优化了改性条件,在浓盐酸与无水乙醇体积比为1∶9条件下,将川陈皮素加热回流反应24 h,可以得到纯度大于97%的5-去甲基川陈皮素。     

不同干燥方式对陈皮中7种黄酮含量及抗氧化性的影响

目的 考察不同干燥方式对广陈皮和普通陈皮中7种黄酮含量及其抗氧化性的影响。方法 对广东新会茶枝柑和福建芦柑果皮采用3种干燥方式(自然晾晒、热风烘干、冷冻干燥)制备陈皮, 利用高效液相色谱法测试样品中7种黄酮(橙皮苷、香蜂草苷、甜橙黄酮、去甲基川陈皮素、3,5,6,7,8,3’,4’-七甲氧基黄酮、川陈皮素、橘皮素)的含量, 采用二苯苦味酰肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)、2,2’-联氮-二-(3-乙基-苯并噻唑啉-6-磺酸)[2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid), ABTS]、OH自由基清除活性和还原能力测试样品的抗氧化性。结果 对于本研究的两种陈皮, 热风烘干和自然晾晒陈皮的黄酮含量较高; 热风烘干陈皮的抗氧化性最高, 其次是自然晾晒, 且OH自由基清除活性较其他方法更能真实地反映陈皮黄酮的抗氧化性。结论 热风烘干制备陈皮具有较高的黄酮含量和抗氧化性。研究结果可为区分了解不同干燥方式制备陈皮的品质提供依据, 同时为陈皮干燥方式的选择提供参考。     

广陈皮促消化功能物质基础的研究

为探究传统药食两用资源广陈皮的促消化功能物质基础,采用国家卫生部颁布的"促进消化功能检验方法",跟踪广陈皮提取物不同极性部位对鼠促消化作用,指导活性物质的分离纯化和结构鉴定,验证各单体化合物对鼠促消化作用。结果表明,广陈皮中的乙酸乙酯提取物为促消化活性最强部分,从中分离纯化并鉴定了橙皮苷、川陈皮素和橘皮素3个黄酮类化合物。橙皮苷对大鼠胃液量、胃蛋白酶排出量、胃蛋白酶活力的影响与空白对照无显著性差异,川陈皮素、橘皮素及三者组合(橙皮苷∶橘皮素∶川陈皮素=5∶1∶1,质量比)可显著促进胃液、胃蛋白酶的排出,提高胃蛋白酶活力,增强消化功能。其中,橙橘川组的蛋白酶排出量为(239.61±43.39)U/h,显著高于空白对照组(145.64±33.29)U/h,小肠墨汁推进率(61.94±6.01)%较空白组提高了35.54%。结果表明,广陈皮中含量最高的成分——橙皮苷的促消化活性较弱,多甲氧基黄酮类化合物川陈皮素和橘皮素的促消化活性较强,说明多甲氧基黄酮为广陈皮促消化功能的主要物质基础。     

柑、橘、橙果皮中天然羟基化多甲氧基黄酮的转化率研究

柑橘果皮中的多甲氧基黄酮,尤其是羟基化多甲氧基黄酮具有抗癌、抗炎等一系列显著的生物活性。本文建立一种有效分析柑橘种属羟基化多甲氧基黄酮的方法,对柑(芦柑)、橘(柳叶橘)、橙(赣南脐橙)果皮采用乙酸乙酯回流提取2次,继而分别用正己烷-甲醇(体积比1∶1)分别萃取,减压浓缩成浸膏,进行梯度洗脱的HPLC-UV分析。结果表明,柑橘种属果皮中多甲氧基黄酮主要集中于甲醇层,约占柑皮鲜重0.54%,橘皮、橙皮中含量略低。柑橘种属果皮中羟基化多甲氧基黄酮主要发生在黄酮母核A环5位上。柑皮中羟基化多甲氧基黄酮主要为5-羟基川陈皮素(141.39±1.84)μg/g、5-羟基橘皮素(19.67±1.24)μg/g、5-羟基甜橙黄酮(13.98±2.24)μg/g、5-羟基-3,6,7,8,3′,4′-六甲氧基黄酮(4.95±1.08)μg/g,转化率分别达到15.28%,3.87%,39.12%和17.15%,转化水平显著高于橘皮和橙皮。橘皮中未检出5-羟基甜橙黄酮,橙皮中未检出5-羟基橘皮素。研究(羟基化)多甲氧基黄酮提取、检测及天然羟基化转化率,对深入挖掘柑橘原料资源,开发预防慢性疾病产品具有重要指导意义。     

磷化氢熏蒸对陈皮品质的影响

为研究磷化氢熏蒸对陈皮品质的影响,测试了陈皮(大红皮和青皮)中总黄酮、橙皮苷、甜橙黄酮、川皮苷、橘皮素以及陈皮挥发油中的D-柠檬烯、γ-松油烯、2-(甲氨基)苯甲酸甲酯等化合物含量在磷化氢熏蒸前后的变化,结果表明,在磷化氢浓度为2.1 g/m~3时,上述化合物含量变化不显著;在磷化氢浓度为8.4 g/m~3时,大红皮中D-柠檬烯含量显著减少,2-(甲氨基)苯甲酸甲酯显著增加,青皮中总黄酮、橙皮苷、D-柠檬烯含量显著减少,2-(甲氨基)苯甲酸甲酯显著增加。     

新会柑胎仔与青皮、陈皮的黄酮含量分析与比较

采用高效液相色谱(High Performance Liquid Chromatography,HPLC)与分光光度法两种检测手段,分析和比较了新会柑胎仔、1年青皮、5年陈皮、10年陈皮4种样品的黄酮苷类和多甲氧基黄酮类化合物的含量。HPLC检测结果表明,新会柑胎仔的橙皮苷含量高达12.8%,远远高于其他3种样品。同时,新会柑胎仔的橘皮素(0.3%)和川陈皮素含量(0.6%)也略高于其他3种样品。分光光度法的检测结果显示,4种样品中黄酮苷类成分含量由高到低分别是柑胎仔(9.5%),10年陈皮(6.1%),5年陈皮(5.2%),1年青皮(4.9%);多甲氧基黄酮类成分含量由高到低分别是柑胎仔(2.5%),1年青皮(1.9%),5年陈皮(1.7%),10年陈皮(1.6%)。两种检测结果均表明柑胎仔中黄酮含量远远高于青皮或陈皮,在功能食品的研究和开发中具有应用潜力。     

野生白木香叶乙酸乙酯提取物化学成分的分离鉴定

为探究沉香茶系列保健食品的主要原料白木香(Aquilaria sinensis)叶片药理活性的物质基础,本研究通过含70%丙酮的水溶液渗漉提取、石油醚和乙酸乙酯多次萃取后,运用硅胶柱色谱、Sephadex LH-20凝胶柱色谱和制备高效液相色谱等技术从野生白木香叶片乙酸乙酯萃取部位中分离纯化得到7个化合物,根据MS、NMR等波谱数据分别鉴定其中6个化合物为麦角甾醇(1)、槲皮素-3-O-β-D-吡喃半乳糖苷(2)、豆甾醇(3)、5,4'-二羟基-7-甲氧基二氢黄酮(4)、谷甾醇(5)、槲皮素-3-O-β-D-吡喃葡萄糖苷(6)。6个化合物按照结构可分为甾醇及其衍生物类(3个),黄酮苷类(2个)和二氢黄酮类(1个)。其中化合物1-2为首次在瑞香科植物中分离得到,化合物4是在瑞香科沉香属植物中首次分离得到的二氢黄酮类化合物,并首次报道了该化合物的2D NMR数据及归属。     

23个柑橘品种果实油胞层类黄酮组分鉴定与抗氧化活性研究

选取23个柑橘品种果实油胞层,采用高分辨质谱等技术鉴定其主要类黄酮组分,研究其抗氧化活性。结果表明:柑橘果实油胞层的总酚含量在4.38～21.10 mg GAE/g DW之间。柑橘果实油胞层综合抗氧化能力最强,最弱的品种分别为“本地早”“爱媛38”。从23种柑橘果实油胞层中共鉴定出53种类黄酮,包括5种黄酮-C-糖苷,4种黄酮-O-糖苷,12种黄烷酮-O-糖苷,32种多甲氧基黄酮(PMFs),有2种类黄酮为首次在柑橘中被鉴定出,分别是根皮素-3’,5’-二-C-葡萄糖苷和槲皮-3-O-鼠李糖基己糖苷。油胞层中的黄烷酮类化合物主要为圣草次苷、新圣草次苷、柚皮芸香苷、橙皮苷和香蜂草苷,PMFs主要为川陈皮素、橘皮素、5HPMF和甜橙黄酮。本文进一步挖掘并补充不同柑橘品种组分和抗氧化活性信息,为活性研究提供理论依据,也为消费提供科学指导。     

HPLC法测定“长林”系列油茶籽种仁中两个主要黄酮苷含量

目的：建立油茶籽种仁中2个主要黄酮苷：山奈酚-3-O-{β-D-吡喃葡萄糖基-(1→2)-[α-L-吡喃鼠李糖基-(1→6)]-β-D-吡喃葡萄糖苷}(Ⅰ)和山奈酚-3-O-{β-D-吡喃木糖基-(1→2)-[α-L-吡喃鼠李糖基-(1→6)]-β-D-吡喃葡萄糖苷}(Ⅱ)的高效液相色谱含量测定方法,并用于“长林”系列油茶品种间黄酮类成分含量评价;优化油茶籽种仁中2个主要黄酮苷的提取工艺。方法：采用Venusil MP C₁₈ (4.6mm×250mm,5μm) 色谱柱,乙腈-0.1%磷酸水溶液作为流动相,线性梯度洗脱,流速1.0 mL.min_-1;检测波长220 nm,柱温25℃;采用L₉(3₄)正交试验设计,比较甲醇浓度、料液比、提取温度、提取时间对黄酮苷提取效率的影响。结果：以80%甲醇按照料液比1:50(g/mL),在80℃条件下回流提取60min,2个黄酮苷的得率最高,11个品种油茶籽种仁中黄酮苷Ⅰ质量分数为1.28%~2.98%,黄酮苷Ⅱ质量分数为1.42%~2.37%。结论：该含量测定方法专属性强、准确度高、重复性好,可用于测定不同品种油茶籽种仁中主要黄酮苷的含量。     

杂环氨基酸酯的合成与表征

以三种杂环结构的氨基酸、二氯亚砜、甲醇/乙醇为原料,合成L-色氨酸甲/乙酯盐酸盐、L-组氨酸甲酯盐酸盐以及L-脯氨酸甲/乙酯盐酸盐,产率较高,并通过核磁共振谱、红外光谱、元素分析对其盐酸盐进行了结构表征,所得到的氨基酸酯盐酸盐分别用饱和碳酸钠溶液中和、二氯甲烷萃取,最终得到了三类杂环氨基酸酯。     

RADIOACTIVE METHIONINE INCORPORATION INTO PEPTIDE CHAINS BY ENZYMATIC MODIFICATION

Enzymatic peptide modification was carried out using L-methionine-S-methyl-¹⁴C methyl ester hydrochloride and L-^3H methionine ethyl ester hydrochloride in the reaction mixture. The experimental results revealed that part of the L-methionine-S-methyl-¹⁴ C methyl ester was bound as methionine to enzymatic hydrolysates of casein and serum albumin in the presence of α-chymotrypsin, interestingly in the highest molecular weight fraction of the product protein. A maximum curve was found to describe the relation between α -chymotrypsin-induced incorporation of methionine and the ratio of L-^3H methionine ethyl ester to protein hydrolysate. The covalent nature of amino acid incorporation was supported by SDS polyacrylamide gel electrophoresis in the presence of urea. The isoelectric focusing patterns of the products indicate that transpeptidation plays an essential role in the EPM reaction. These experimental findings suggest that enzymatic peptide modification is a suitable method for the production of proteins of designed amino acid composition.     

Determination of synergistic effects of polymethoxylated flavone extracts of Jinchen orange peels (<Emphasis Type="Italic">Citrus Sinensis</Emphasis> Osberk) with amino acids and organic acids using chemiluminescence

Antioxidant capacity of polymethoxylated flavones (PMFs) extracts of Jinchen orange peels (Citrus sinensis Osberk) was evaluated by using a chemiluminescence (CL) method in vitro. PMFs were isolated using a procedure that produced a consistent mixture of PMFs both in identity and in proportion. The mixture mainly consisted of nobiletin (55.9%), sinensetin (15.2%), heptamethoxyflavone (13.2%), and tangeretin (8.1%). The antioxidative effects of PMFs and the main individual flavones in PMFs as well as synergistic effects of PMFs with organic acids (citric acid, oxalic acid, and malic acid) and amino acids (alanine, lysine, serine, and threonine) on superoxide anion were determined by the pyrogallol–luminol system. The results showed that the inhibition by PMFs on superoxide radical was stronger than that of the individual flavones. Mixtures of citric acid and lysine with PMFs had higher synergistic effects than any other tested mixtures, and the optimum concentrations were exhibited by combinations of 7 μg/mL PMFs + 120 μg/mL citric acid (1.2, p < 0.05) and 7 μg/mL PMFs + 42 μg/mL lysine (1.1, p < 0.05). For the other amino acids and organic acids, the values of the measured inhibition effect (% MIE) of the mixtures with PMFs were not significantly different from their expected inhibition effect (% EIE), which did not show synergism.     

Effect of rosehip (Rosa canina L.) phytochemicals on stable free radicals and human cancer cells

BACKGROUND: The commercial development of plants as sources of antioxidants that can be used to enhance the properties of foods, for nutritional purposes and preservation as well as for prevention of oxidation‐related diseases, is currently of major interest. Rosehip (Rosa canina L.) is a rich source of vitamin C and polyphenols. RESULTS: Phytochemicals in rosehip tea were separated into three fractions: Fr1 (vitamin C, 39.17 mg kg^?1), Fr2 (flavonoids, 451.05 µg kg^?1) and Fr3 (phenolic acids, 504.69 µg kg^?1). Quercetin and ellagic acid were the most abundant polyphenolic compounds. Rosehip fractions, primarily rosehip flavonoids (EC₅₀ = 49 mg L^?1), showed high antioxidant activity towards 2,2‐diphenyl‐1‐picrylhydrazyl radicals (DPPH^?). Cell growth effects of rosehip fractions were assessed in HeLa, MCF7 and HT‐29 cell lines, with the lowest IC₅₀ values being determined for rosehip flavonoids, (80.63, 248.03 and 363.95 mg L^?1 respectively). However, the vitamin C fraction did not inhibit the growth of tested tumour cells. CONCLUSION: The results of this study confirm that vitamin C and flavonoids are responsible for the antioxidant activity of rosehip tea, while only polyphenols contribute to its antiproliferative activity. Copyright © 2011 Society of Chemical Industry     

24 kDa Trypsin: A predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus)

Trypsin was purified to homogeneity from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) through ammonium sulphate fractionation and a series of chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. It was purified to 47.6-fold with a yield of 12.7%. Based on native-PAGE, the purified trypsin showed a single band. The molecular weight of purified trypsin was estimated as 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature for N^α-p-tosyl-l-arginine methyl ester hydrochloride (TAME) hydrolysis were 8.0 and 60 °C, respectively. Trypsin was stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. Trypsin was stabilized by calcium ion. The trypsin activity was strongly inhibited by soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone and partially inhibited by ethylenediaminetetraacetic acid. Activity decreased continuously as NaCl concentration (0–30%) increased. Apparent K_m value of trypsin was 0.3 mM and K_cat value was 92.1 S⁻¹ for TAME. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGYECQAHSQPPTVSLNA, which is highly homologous with trypsins from other species of fish.     

不同晚熟柑橘中酚类物质的含量检测及分析

采用超高效液相色谱(ultra-performance liquid chromatography,UPLC)对9种晚熟柑橘(沃柑、春见、大雅柑、不知火、默科特、红肉脐橙、伦晚脐橙、红翠2号和塔罗科血橙)中的主要酚类物质(13种类黄酮和7种酚酸)进行测定,分析比较不同品种柑橘果皮、果肉和果汁(柑橘原汁)中酚类物质种类和含量的差异。结果表明:9个柑橘品种的果皮、果肉、果汁中,类黄酮均以橙皮苷为主,酚酸以阿魏酸为主,且果皮中多甲氧基黄酮含量丰富。在9个柑橘品种果汁中,春见的橙皮苷、咖啡酸含量最高。果肉中,沃柑中阿魏酸、咖啡酸、芥子酸含量最高,塔罗科血橙的橙皮苷、对香豆酸含量最高。果皮中,默科特的芸香柚皮苷、川皮苷、阿魏酸含量最高。杂柑果皮中咖啡酸、阿魏酸及川皮苷含量高于橙类。晚熟柑橘含有丰富的酚类物质,并呈现显著的多样性。     

Polymethoxy flavones are responsible for the anti-inflammatory activity of citrus fruit peel

In traditional Chinese medicine, dried citrus fruit peels are widely used as remedies to alleviate some respiratory inflammatory syndromes and are considered as potential sources of anti-inflammatory components. Seven citrus fruits were selected for this study. We determined the inhibitory ability of citrus peel extracts on the production of pro-inflammatory mediators, prostaglandin E₂ (PGE₂) and nitric oxide (NO), in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Among the tested citrus peels, Ponkan (Citrus reticulata Blanco) and Tonkan (Citrus tankan Hayata) deserve special attention due to their outstanding inhibitory effect on PGE₂ and NO secretion. We have also examined the composition, flavanone glycosides and polymethoxy flavones. The polymethoxy flavone content, especially nobiletin, appears to correlate well with the anti-inflammatory activities of certain citrus peel extracts. Thus, our results suggest that polymethoxy flavones contribute crucially to the anti-inflammatory activity of citrus peels.     

The solubilisation and hydrolysis of spinach cell wall polysaccharides in gastric and pancreatic fluids

The aim of this work was to investigate the fate, in the upper gut of a monogastric animal, of polymers bound within plant cell walls. Uniformly and specifically ¹⁴C-labelled spinach cell walls were incubated in artificial body fluids with and without pepsin or pancreatin. In the absence of enzymes, artificial pancreatic juice (pH 8.8) at 37°C hydrolysed the methyl ester groups of wall-bound pectins [half-life (t^1/2) 25 h]; the O-acetyl ester groups of cell wall polysaccharides were much more stable (t ^1/2 84 h). In contrast, artificial gastric juice (pH 1.85) hydrolysed wall-bound acetyl groups more rapidly (t ^1/2 ～ 24 h) than methyl ester groups (t ^1/2 ～ 350 h). Thus, a proportion of the methyl and acetyl groups of plant cell wall polymers will be released in the upper gut as methanol and acetic acid, raising the question of whether these groups should be included within the definition of dietary fibre. The artificial body fluids also caused limited solubilisation of wall polymers but no hydrolysis to mono- or oligosaccharides. Neither pepsin nor pancreatin promoted the hydrolysis of methyl ester or acetyl groups. The small amounts of [¹⁴C]protein present in the cell wall preparations were hydrolysed by pepsin or pancreatin to yield amino acids and oligopeptides; however, the major polysaccharides of spinach cell walls were neither degraded to low-molecular-weight products nor solubilised by these enzymes.     

Enzymatic and chemical oxidation of trihydroxylated phenols

Gallic acid, pyrogallol and gallic acid methyl ester were kinetically characterised as substrates of polyphenoloxidase (PPO) and peroxidase (POD) and their pro- and anti-oxidant capacities were studied. The data obtained are correlated with the chemical shifts of the carbons supporting the vicinal hydroxyls obtained by ¹³C NMR. Pyrogallol showed the highest catalytic constant for both PPO and POD, reflecting its low δ₂ value. This also implies that both enzymes have a high Michaelis constant for pyrogallol.     

Insect attractant and oviposition enhancing activity of hexadecanoic acid ester derivatives for monitoring and trapping Caryedon serratus

This paper describes behavioural changes elicited in Caryedon serratus by some synthesized hexadecanoic acid ester derivatives, which can be effectively used for monitoring pest populations thereby reducing the risk of pests becoming established in stores. Six hexadecanoic acid ester derivatives were synthesized. Choice and no-choice experiments with different concentrations (0.1, 0.5 and 1.0 mg l⁻¹) of derivatives against C. serratus revealed significant (p < 0.001) oviposition activity for five Hexadecanoic acid ester derivatives. In contrast, 1.0 mg l⁻¹ benzoyl hexadecanoate reduced oviposition (4.7 ± 0.6) significantly (p < 0.001) as compared with control. In Y-tube olfactometer bioassays performed under laboratory conditions, synthesized derivatives elicited significant (p < 0.001) attractiveness (70–98%) in female C. serratus. Additionally, trap experiments performed in mini-stores also support highly significant (p < 0.001) attractiveness of 3-methylphenyl hexadecanoate (0.1 mg per 10 ml) and 4-bromophenyl hexadecanoate (1.0 mg per 10 ml). Conversely, benzoyl hexadecanoate significantly attracted male C. serratus. This study established the dose specific application of hexadecanoic ester derivatives in traps for effective management of this groundnut pest. The specificity of synthesized ester derivatives in attracting both sexes differently also indicates their probable resemblance to pheromone components of C. serratus.