原花青素提取及微胶囊化研究

作为葡萄加工的副产物,葡萄籽中富含葡萄籽油和低聚原花青素。作者利用超临界二氧化碳萃取葡萄籽后所得的残渣为原料,以含有0.8%醋酸的乙醇溶液为提取剂来提取其中的原花青素,在55℃条件下进行两次重复提取,葡萄籽残渣与提取液的比例控制在1∶8(W/V),每次提取60 min,原料中原花青素的提取率可以达到98.2%;为提高产品的贮藏稳定性,还对以阿拉伯胶与麦芽糊精组合作为原花青素微胶囊壁材来进行微胶囊化的工艺进行研究,结果表明在阿拉伯胶占壁材40%、芯壁材比为3∶7,混合液中固形物含量为20%的条件下,经喷雾干燥后所得原花青素的产率为88.84%,微胶囊化效率达到99.2%。检测结果表明,原花青素紫外吸收光谱在微胶囊化前后没有变化,而其贮藏稳定性得到提高。     

树莓种子原花青素的提取分离工艺研究

本文对树莓种子原花青素的提取及分离的工艺条件进行了研究和探讨。结果表明:水对原花青素的提取效果较好。用10倍于树莓种子重量的水(pH 8)于80℃提取三次,每次90 min,原花青素的提取效果最佳。提取液浓缩后,用NKA树脂吸附,当上样液pH值为7时,以50%乙醇溶液洗脱,分离出的原花青素含量最高,达23.66%,且产品得率也最大,达11.0%。     

肉桂高聚原花青素的氢化降解工艺研究

低聚原花青素具有显著的生物活性,高聚体由于空间位阻的影响,活性受到抑制。肉桂在41种食品中原花青素含量位居第一,但高聚体占50％以上。为了充分利用肉桂原花青素资源,采用氢化降解法将肉桂高聚原花青素降解为低聚体。以10％钯碳做催化剂,高压氢气参加反应,考察温度、压强、反应时间和催化剂用量对降解效果的影响。结果表明,平均聚合度为8．2的肉桂高聚原花青素,在100℃,压强3．5MPa,反应200min,催化剂用量0．2g/100mL的条件下,平均聚合度降为2．68。降解产物经HPLC分析表明,此工艺确实实现了肉桂高聚原花青素的降解。     

山葡萄籽中原花青素的提取

研究了提取溶剂、温度、时间、料液比4个因素对山葡萄籽中原花青素得率的影响.确定最佳提取工艺以70%丙酮为提取剂,在60℃条件下,提取120 min,料液比为17,原花青素的得率为2.31%     

“黑美人”马铃薯中原花青素抗氧化性研究

在超声辅助下从"黑美人"马铃薯中提取原花青素,提取浓缩液经AB-8大孔吸附树脂填充柱纯化后,所得产物中原花青素含量为93.59%,得率为4.3%。抗氧化性研究表明,其对DPPH·自由基、·OH自由基和O~(-·)_2自由基具有显著的清除效果,清除率与其浓度成正相关,且不输于V_C,尤其是对·OH自由基和O~(-·)_2自由基的清除作用明显高于V_C。表明"黑美人"马铃薯原花青素有较强的抗氧化性,这为进一步开发利用"黑美人"马铃薯提供必要的理论支持。     

落叶松树皮原花青素提取工艺的研究

本文考察了落叶松树皮原花青素的料液比、乙醇浓度、提取温度、超声时间对原花青素提取效果的影响。采用正交实验,对提取工艺进行了优化。结果表明各因素对原花青素的提取效果的影响程度为提取温度>提取时间>乙醇浓度>料液比。从而确定落叶松树皮中原花青素的最佳工艺条件为:提取温度为35℃,提取时间为0.5 h,乙醇浓度为50%,料液比为1∶15(g/mL)。     

葡萄籽中原花青素的提取及应用现状

原花青素（proanthocyanidins,PC)是目前国际上公认的清除人体内自由基最有效的天然抗氧化剂,广泛分布于多种天然植物中。阐述了葡萄废弃物中原花青素的功能,分析了其应用开发现状。结合常用的提取方法,并综合国内外关于原花青素的研究进展,对葡萄籽中原花青素提取的工艺参数进行优化,从而得出葡萄籽中原花青素最优提取方案。以期为葡萄籽的全面利用和原花青素的工业化生产提供科学依据,使原花青素拥有更广泛的应用。     

锁阳原花青素对葡萄糖/甘氨酸模拟体系糖基化终产物的抑制效果

为探究锁阳原花青素对葡萄糖/甘氨酸模拟体系中形成的晚期糖基化终产物(advanced glycation end-product,AGEs)的抑制效果。本实验考察了不同温度,不同反应时间下锁阳原花青素对体系中AGEs抑制的影响,同时探讨了多种金属离子对抑制效果的作用。结果表明:在80℃下加热75 min,锁阳原花青素的质量浓度为1 mg/mL时,对AGEs的抑制效果可达85.73%±1.57%,而100℃下加热30 min时抑制率达到74.01%±1.45%。当加入金属离子(Al³⁺、Cu²⁺、Fe²⁺、Mg²⁺、Ca²⁺、Zn²⁺)时,对AGEs的形成既有抑制作用也有促进作用;与仅有锁阳原花青素存在时相比,金属离子在低浓度时减弱了锁阳原花青素对AGEs的抑制作用,高浓度差异显著。此结果可为天然AGEs抑制剂的开发和在食品中应用提供参考价值。     

植物原花青素生物合成及调控研究进展

原花青素是通过类黄酮途径生成的一类多酚类化合物。原花青素具有重要的生物学功能,不仅是植物应对生物和非生物胁迫的一种重要防御手段,还能影响植物外观、风味和品质,因此原花青素合成途径一直是作物性状改良的研究热点。该文主要在模式植物拟南芥研究的基础上,综述了原花青素生物合成研究的最新进展,讨论了原花青素遗传工程应用前景和主要限制因素,旨在为进一步开展原花青素的研究和应用提供参考。     

酶辅助提取莲房原花青素工艺及其抗氧化活性研究

主要用纤维素酶和果胶酶对莲房组织进行酶解,以提高莲房原花青素提取率。采用四因素三水平正交实验对酶解时间、加酶量和酶解温度等提取工艺参数进行优化,获得了最佳工艺参数为纤维素酶添加量为0.7%,果胶酶添加量0.1%,酶解温度55℃,酶解时间2.5h,优化后的提取工艺与直接醇提法相比,能将莲房原花青素的提取率提高约48%。在此基础上采用DPPH法进行了抗氧化性的对比,结果表明酶辅助提取和醇提法提取的原花青素有着相同的抗氧化性。     

Paeoniflorin diminishes ConA-induced IL-8 production in primary human hepatic sinusoidal endothelial cells in the involvement of ERK1/2 and Akt phosphorylation

Liver diseases are closely associated with elevated levels of interleukin-8 (IL-8), suggesting the ability to inhibit IL-8 production could enhance the treatment of liver diseases. Paeoniflorin is a major active constituent of dried Paeoniae Radix Alba root (Baishao in Chinese) which is widely used in China to treat liver diseases. We examined the effects and underlying mechanisms of paeoniflorin on IL-8 production in primary human hepatic sinusoidal endothelial cells (HHSECs). Concanavalin A (ConA) at 20 μg/mL produced a 5.2-fold increase in IL-8 mRNA by 8 h, and a 14.2-fold rise in IL-8 levels by 16 h. Inhibition of MEK (ERK kinase) and extracellular signal-regulated kinase (ERK) by PD98059 and U0126, or inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked both ConA-induced IL-8 mRNA expression and IL-8 secretion. Paeoniflorin reduced ConA-induced IL-8 mRNA expression and IL-8 release by 57.9% and 52.8%, respectively, and also decreased ConA-stimulated phosphorylation of ERK1/2 and Akt, suggesting paeoniflorin inhibits IL-8 expression and release by inhibiting the ERK1/2 and Akt pathways. Combining paeoniflorin with U0126 or LY294002 at low doses showed supra-additive inhibition of not only phospho-ERK1/2 and phospho-Akt by 46.4% and 35.0%, but also IL-8 release by 42.4% and 36.1% and IL-8 mRNA expression by 43.5% and 31.8%, respectively. In conclusion, paeoniflorin most likely contributes to the therapy for liver disease by exerting anti-inflammatory effects on HHSECs through blocking IL-8 secretion via downregulation of ERK1/2 and Akt phosphorylation.     

Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides for scintigraphic imaging of CCK receptors

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two (99m)Tc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). (99m)Tc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (approximately 95%) and yielded products with a high specific activity (approximately 700 Ci/mmol) and good stability (approximately 5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 degrees C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized approximately 3% of added (99m)Tc-sCCK8 per confluent well during 2 h at 37 degrees C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize (99m)Tc-nsCCK8. Displacement of (99m)Tc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 degrees C to prevent internalization) revealed 50% inhibitory concentrations (IC(50)) of 8 nM and >1 microM, respectively. CHO-CCK2 cells internalized approximately 25% and approximately 5% of added (99m)Tc-sCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC(50) values for the displacement of (99m)Tc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected (99m)Tc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of (99m)Tc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of (99m)Tc-sCCK8 and -nsCCK8 by CHO-CCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of (99m)Tc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of (99m)Tc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with (99m)Tc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate (99m)Tc-nsCCK8, CCK2 tumors accumulate 2 times more (99m)Tc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more (99m)Tc-sCCK8 than (99m)Tc-nsCCK8. Although accumulation in some nontarget organs was also higher with (99m)Tc-sCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of (99m)Tc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.     

Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

OSBP-related protein 8 (ORP8) regulates plasma and liver tissue lipid levels and interacts with the nucleoporin Nup62

We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum oxysterol-binding protein implicated in cellular lipid homeostasis. We now investigated its action in hepatic cells in vivo and in vitro. Adenoviral overexpression of ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum (-34%, -26%, -37%, respectively) and liver tissue (-40%, -12%, -24%), coinciding with reduction of nuclear (n)SREBP-1 and -2 and mRNA levels of their target genes. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing by RNA interference moderately suppressed or induced the expression of SREBP-1 and SREBP-2 target genes, respectively. In accordance, cholesterol biosynthesis was reduced by ORP8 overexpression and enhanced by ORP8 silencing in [(3)H]acetate pulse-labeling experiments. ORP8, previously shown to bind 25-hydroxycholesterol, was now shown to bind also cholesterol in vitro. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation analyses revealed the nuclear pore component Nup62 as an interaction partner of ORP8. Co-localization of ORP8 and Nup62 at the nuclear envelope was demonstrated by BiFC and confocal immunofluorescence microscopy. Furthermore, the impact of overexpressed ORP8 on nSREBPs and their target mRNAs was inhibited in cells depleted of Nup62. Our results reveal that ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism, and provide clues of an entirely new mode of ORP action.     

mLST8在大肠杆菌中的表达、纯化及鉴定

目的:表达并纯化mLST8蛋白。方法:PCR扩增mLST8的编码cDNA,克隆到pET-28a(+)表达载体,将重组质粒pET-28a-mLST8转化大肠杆菌BL21(DE3)感受态细胞,在IPTG诱导下表达目的蛋白;提取包涵体,用Ni2+亲和层析纯化目的蛋白,稀释和透析相结合进行复性,对复性蛋白进行阴离子交换层析、分子筛层析,将纯的复性mLST8进行肽指纹质谱鉴定和圆二色谱分析。结果:酶切和DNA测序证明pET-28a-mLST8表达质粒构建无误,并在大肠杆菌中得到高效表达;通过Ni2+亲和层析、复性、离子交换层析和分子筛层析获得了较高纯度的复性蛋白,肽指纹质谱鉴定为mLST8;mLST8蛋白的二级结构[α螺旋为18.2%,β折叠为52.3%(其中平行结构为12.1%,反向平行结构为40.2%),β转角为20.7%,无规则卷曲为39.9%]表明其为典型的β折叠结构。结论:在大肠杆菌中表达了重组mLST8蛋白,复性获得了二级结构准确的mLST8,为进一步研究mLST8的晶体结构与功能奠定了基础。     

大鼠肥厚心肌缩短速度和肌凝蛋白重链同功酶谱的变化

用聚丙烯酰胺电泳分离并测定了大鼠左室肌凝蛋白ＡＴＰ酶活性依次降低的同功酶Ｖ_１,Ｖ_２和Ｖ_３的百分比（ＭＩ谱）,从乳头肌力－速度曲线读取心肌最大缩短速度（Ｖ_（ｍａｘ））,观察到：（１）正常大鼠出现增龄性Ｖ_１向Ｖ_３迁移和Ｖ_（ｍａｘ）下降,与８周龄组（Ｓ_０）相比,１６周和２４周龄组（Ｓ_８和Ｓ_（１６））的Ｖ_１／Ｖ_３比,分别下降３８．９％和６１．０％;Ｖ_（ｍａｘ）下降８．３％和１３．３％。（２）高血压肥厚心肌ＭＩ谱的迁移和Ｖ_（ｍａｘ）下降的程度大大超过增龄效应：高血压８周和１６周组（Ｈ_８和Ｈ_（１６））的Ｖ_１／Ｖ_３比值较术前对照Ｓ_０分别下降８４．４％和９３．５％,较同龄假手术对照Ｓ_８和Ｓ_（１６）组也分别低７４．５％和８３．３％,而Ｖ_（ｍａｘ）则比Ｓ_０组下降３３．３％和４８．３％。（３）６组４８只大鼠结果相关分析表明,Ｖ_（ｍａｘ）与Ｖ_１％高度正相关,与Ｖ_３％高度负相关。（Ｐ均小于０．０１）。上述结果提示：高血压肥厚心肌收缩速度明显下降,其主要生化机制似与同功酶谱由Ｖ_１优势向Ｖ_３迁移有关。     

Glycosylation of capsaicinoids with Panax ginseng stimulated by salicylic acid

The efficient production of β-glycosides of capsaicin and 8-nordihydrocapsaicin by cultured cells of Panax ginseng is reported. Capsaicin 4-O-(6-O-β-D-xylopyranosyl)-β-D-glucopyranoside (β-primeveroside, 12%) together with capsaicin 4-O-β-D-glucoside (6%) was isolated from the cell suspension of P. ginseng after one week of incubation with capsaicin. On the other hand, 8-nordihydrocapsaicin was glycosylated to 8-nordihydrocapsaicin 4-O-β-D-glucoside (5%) and 8-nordihydrocapsaicin 4-O-β-primeveroside (9%) by P. ginseng. Pretreatment of the cultured cells with salicylic acid greatly enhanced the glucosylation activity toward capsaicinoids. When 500 μM of salicylic acid was added to the cultures prior to the addition of substrate, capsaicin was converted into capsaicin 4-O-β-D-glucoside (17%) and capsaicin β-primeveroside (21%) and 8-nordihydrocapsaicin was glycosylated to 8-nordihydrocapsaicin 4-O-β-D-glucoside (16%) and 8-nordihydrocapsaicin β-primeveroside (15%).     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Pre-treatment of recombinant mouse MFG-E8 downregulates LPS-induced TNF-α production in macrophages via STAT3-mediated SOCS3 activation

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8(-/-) mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8(-/-) mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8(-/-) mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.