祁白术多酚酶法提取工艺优化及其抗氧化、抑制黑色素合成活性

为考察祁白术多酚(polyphenols from Atractylodes macrocephala Koidz grown in Qimen, AMP)的可利用性,采用响应面分析法优化酶法提取工艺,并研究AMP的抗氧化、抑制黑色素合成活性。结果表明,在料液比1:30 g·mL^-1、酶解时间20 min的条件下,当纤维素酶添加量为1.35%、酶解温度为44℃、pH值为4.7、搅拌转速为670 r·min^-1时,AMP提取量最高,为26.58±0.23 mg·g^-1。统计学分析显示,所选响应面模型拟合较好,优化后的提取工艺条件合理可行。AMP具有较好的抗氧化活性,其总还原力、对1,1-二苯基-2-三硝基苯肼(DPPH)和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)自由基清除作用随其浓度的增加而增强。当AMP浓度为0~0.02 mg·mL^-1时,对B16细胞无毒性作用。与α-MSH模型组相比,AMP显著下调了细胞内酪氨酸酶活性(P<0.05),并呈浓度依赖性地抑制细胞内黑色素的合成(P<0.05);当AMP浓度为0.02 mg·mL^-1时,对细胞内酪氨酸酶活性及黑色素合成抑制率分别为30.11%、43.35%,阳性对照熊果苷(0.1 mg·mL^-1)对细胞内酪氨酸酶活性及黑色素合成的抑制率分别为22.03%、39.77%,表明AMP对黑色素生成的抑制效果强于熊果苷。本研究结果为祁白术多酚的综合开发利用提供了依据。     

猕猴桃皮多酚提取及其对B16细胞黑色素的影响

植物多酚类物质对皮肤增白和美容有着重要的作用,是美容护肤保健品中的主要成分,猕猴桃皮中多酚类物质含量丰富,可成为美容保健品的原料。为了探究猕猴桃皮中多酚类物质的提取及其美白作用,该研究采用深共熔溶剂（Deep-Eutectic Solvents,DESs）提取猕猴桃皮多酚,通过单因素试验和响应面试验确定优化提取工艺,用高效液相色谱分析猕猴桃皮多酚的成分,并研究猕猴桃皮多酚对酪氨酸酶的影响及对B16细胞黑色素的作用机制。结果表明：深共熔溶剂是提取猕猴桃皮多酚的较佳溶剂,提取猕猴桃皮多酚优化工艺为DES-1含水率18%、料液比1:29 g/mL、提取时间22 min、提取温度69 ℃,猕猴桃皮多酚得率55.53 mg/g。猕猴桃皮多酚中的主要成分有(+)-儿茶素、绿原酸、咖啡酸和表儿茶素,并且猕猴桃皮多酚对体外酪氨酸酶活性有抑制作用。此外,当猕猴桃皮多酚浓度为100 μg/mL时对B16细胞内黑色素生成有明显的抑制作用,能使细胞内黑色素相对含量降为75.08%,细胞内酪氨酸酶活性降至69.45%,且优于阳性对照组曲酸（P<0.05）。结果表明,猕猴桃皮多酚可作为一种天然美白剂应用于化妆品及其他保健品中。     

动态高压微射流对乌贼墨黑色素物理性质及微观结构的影响

为改善乌贼墨黑色素在食品中的应用性能,以虎斑乌贼(Sepia pharaonis)墨囊为原材料,采用碱性蛋白酶酶解制备乌贼墨黑色素,研究不同动态高压微射流(DHPM)(0、40、80、120、160、200 MPa)均质对其物理性质的影响,并采用红外光谱、扫描电子显微镜分析其微观结构。结果表明,DHPM处理使乌贼墨黑色素聚集体解聚,浊度增加、平均粒径减小,粒度分布更加集中和细微化,同时改善了其在去离子水和生理盐水中的溶解性;在压力作用下表现为粘度、空间色度值均有所增大;差示扫描量热法(DSC)分析表明,乌贼墨黑色素在120 MPa下热变性温度为90.70℃,焓变值为0.45 J·g^-1,且该压力下其贮藏稳定性最好;红外光谱分析表明,在特定压力处理范围内,DHPM处理使乌贼墨黑色素分子间形成了较强的氢键;扫描电镜观察发现样品颗粒在120 MPa处理后颗粒尺寸最小。因此,DHPM改性技术改善了乌贼墨黑色素的物理性质和微观结构,为其在食品工业中的应用提供了理论依据。     

生姜提取物对LPS诱导RAW 264.7细胞炎症反应的干预作用研究

以新鲜生姜为原料制备不同温度干燥的生姜提取物,在LPS诱导RAW 264.7细胞炎症模型上研究生姜提取物的抗炎活性及机理,明确提取物中主要活性成分。采用冷冻干燥、烘箱60℃干燥、烘箱90℃干燥3种方式对新鲜生姜进行干燥,用70%乙醇水溶液超声提取得到3种生姜提取物。利用体外抗氧化实验(TEAC、 FRAP和DPPH)比较3种提取物的抗氧化活性,在LPS诱导RAW264.7细胞炎症模型上研究3种生姜提取物对细胞NO、 IL-6、 IL-1β和PGE2分泌、炎症相关基因以及NFκB通路蛋白表达的影响,最后利用HPLC-MS明确3种提取物中主要活性成分的差异。结果显示,生姜提取物表现出抗氧化活性,且可以通过调控NFκB通路抑制LPS诱导RAW264.7细胞促炎因子的分泌和炎症相关基因表达。随着干燥温度的升高,生姜提取物的抗氧化和抗炎活性得到显著提高,主要得益于生姜中姜酚向姜烯酚的转变。     

鸡的酪氨酸酶基因5''''调控区的单链构象多态性分析

酪氨酸酶(byrosinase，TYR)是动物黑色素合成的关键酶。对鸡TYR基因上游调控区序列-641～-2125bp进行了单链构象多态性(SSCP)分析，发现在该区域内存在3个单核苷酸多态(SNP)位点，并分析了SNPS位点与中国农业大学资源群体亲代(P)和F2代个体黑色素性状的相关性。结果显示TYR基因调控区SNPS位点与鸡的胫色和皮肤色显著相关。     

响应面法优化超声波辅助灵芝黑色素提取工艺

为探究超声波辅助法提取灵芝天然黑色素的最佳工艺条件,本试验以灵芝子实体粉为原材料,以黑色素溶液吸光度为响应值,分别考察Na OH浓度、料液比、超声时间和超声功率4个因素对提取效果的影响,并在单因素试验基础上,通过Box-Behnken响应面分析法优化灵芝天然黑色素的超声波辅助提取工艺。结果表明,响应面法合理可行,最佳提取条件为Na OH浓度1 mol·L~(-1)、料液比1∶39.7g·m L~(-1)、超声功率为162 W和超声时间49.8 min,在此条件下灵芝黑色素吸光度为0.782,与预测值0.783相近。本试验从多角度优化超声波辅助提取灵芝黑色素工艺,为灵芝黑色素的进一步加工提供了理论依据。     

粗毛纤孔菌产胞外黑色素发酵条件优化及其抗氧化活性研究

为提高天然黑色素的产量,本研究通过探索碳氮源、转速、pH、温度等单因素对粗毛纤孔菌产黑色素的影响,并利用正交试验筛选产胞外黑色素的最适发酵培养基配方和发酵条件。结果表明,优化后的最佳发酵培养基配方为甘露醇20 g·L^-1、牛肉浸膏5 g·L^-1、碳氮比4∶1、维生素B₁ 10 mg·L^-1,最适发酵条件为发酵温度25℃、发酵初始pH值6、转速180 r·min^-1、发酵时间10 d。在此培养条件下,粗毛纤孔菌胞外黑色素(IHEM)含量高达3.29 g·L^-1,较优化前提高了17.32倍。IHEM体外化学抗氧化活性检测结果显示,IHEM具有良好的抗氧化活性,其对ABTS自由基的清除效果较佳,半最大效应浓度(EC₅₀)为0.019 mg·mL^-1。本研究结果为深层发酵高产黑色素的开发提供了技术支持。     

胨冻样芽孢杆菌(Bacillus mucilaginosus)对烟草灰霉病菌(Botrytis cinerea)的抑制作用

本文首次报道胨冻样芽孢杆菌XDB1和D4B1对烟草灰霉病菌(Botrytis cinerea)的抑制作用。实验表明,XDB1和D4B1的发酵原液和无细胞提取物具有相同的抑菌效果,而经121℃处理30分钟后,抑菌活性丧失。D481菌株的抑菌活性高于XDB1,钾长石作为基质时的活性高于土壤矿物。但是,XDB1和D481对立枯丝核病菌(R..solani)、西瓜尖孢镰刀菌(F.oxysporum)、腐霉(Pythium sp)、小麦全蚀病菌(G.graminis)、烟炭疽病菌(C.gloeosporides)、烟草赤星病菌(A.alternata)、烟草黑胫病菌(P.nicotianae、串珠镰刀病菌(F.moniliforme)和水稻稻瘟病菌(P.grisea)无抑制作用。     

小飞蓬的铜毒害和抗性机制研究

对高铜污染区的小飞蓬进行盆栽实验,在不同Cu浓度处理下,对小飞蓬的电导率、叶绿素以及蛋白质含量进行测定。结果表明:电导率随着Cu浓度的提高而增大,而叶绿素和蛋白质含量先微增后持续减少,这说明Cu损害了小飞蓬的细胞膜,并影响了小飞蓬的光合作用和蛋白质的合成。对小飞蓬的抗性机制研究表明,当土壤Cu浓度升高时,小飞蓬根部Cu含量随着增加,相关系数为0.974**;而地上部分Cu含量的上升并不明显;转移到地上部分的Cu随着土壤铜含量的提高,亚细胞组分铜的总量增加;其中组分F1(细胞壁以及未破碎残渣)、F3(线粒体部分)、F4(核蛋白和可溶性组分)中Cu含量也呈现增加趋势,相关系数为0.992**、0.973**、0.995**,呈现极显著的正相关性;以F1所占比例最高,约为34.7%～40.9%,F4次之,约为26.8%～32.3%。随着土壤Cu浓度的提高,Cu在根部的存在形态中以醋酸提取态为主,其次为盐酸提取态;地上部分的Cu提取态中活性较低的醋酸提取态和盐酸提取态所占比例提高,活性较高的氯化钠提取态和乙醇提取态下降,水提取态变化幅度不是很明显,但总体上以氯化钠提取态和乙醇提取态为主;抗氧化酶系统SOD、POD、CAT在Cu浓度增加时,活性增强,其中CAT、POD与Cu浓度之间的相关系数为0.940*、0.924*,呈现显著的正相关性,这3种酶组成的清除自由基系统对小飞蓬的耐Cu性有很大的影响。     

枣果皮中酚类物质提取工艺优化及抗氧化活性分析

为提高枣果皮中的酚类物质的提取效率,该文以马牙枣为试验材料,对枣果皮中酚类物质提取条件进行了优化,同时通过测定酚类物质清除2,2’-二苯基-1-间三硝苯基联肼(DPPH),2,2'-连氮基双(3-乙基苯并噻唑啉)-6-磺酸(ABTS)自由基和铁还原能力,分析了枣果皮提取物中酚类物质的抗氧化活性。结果显示,枣果皮中酚类物质提取的最优条件是采用70%(V/V)的甲醇、在40℃下,以1∶60的料液比浸提4h,重复浸提2次。枣果皮中酚类物质具有很强的抗氧化能力,与合成抗氧化剂2,6-二叔丁基对甲酚(BHT)相比,虽然果皮中酚类物质清除ABTS·+自由基能力略低于BHT,但其清除DPPH自由基和铁还原能力与之相当。研究结果表明枣果皮中酚类物质具有很强的抗氧化能力,在一定程度上可以取代合成抗氧化剂BHT,应用于食品加工业。     

鱿鱼墨黑色素提取物对肿瘤细胞的抑制作用

为探索鱿鱼墨黑色素提取物(SIME)对体外培养的肿瘤细胞的抑制效应,通过四甲基偶氮唑盐(MTT)法和划痕试验比较SIME对人结肠癌细胞HCT-116、宫颈癌细胞Hela、肝癌细胞Hep G2、乳腺癌细胞MCF-7和前列腺癌细胞PC-3增殖及细胞迁移能力的影响。结果表明,SIME对肿瘤细胞形态有不同程度的影响,对肿瘤细胞增殖有浓度依赖性抑制作用,并具有抑制肿瘤细胞非定向迁移的作用。SIME对HCT-116和PC-3细胞增殖的抑制作用较好,72 h时两者半数抑制浓度IC50分别为0.144mg·m L~(-1)和0.485 mg·m L~(-1)。由此可见,SIME具有较强的抗肿瘤活性,该研究结果为鱿鱼墨高值化利用提供了一定的理论依据。     

Antioxidant and antimelanogenic activities of polyamine conjugates from corn bran and related hydroxycinnamic acids

The antioxidant activity of three major polyamine conjugates, N,N'-dicoumaroyl-putrescine (DCP), N-p-coumaroyl-N'-feruloylputrescine (CFP), and N,N'-diferuloyl-putrescine (DFP) isolated from corn bran, and their related hydroxycinnamic acids, p-coumaric acid and ferulic acid, were evaluated by three antioxidant in vitro assay systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide and hydroxyl radicals generated by enzymatic and nonenzymatic reactions. Additionally, five phenolic compounds were evaluated for melanogenesis inhibitory activity using mushroom tyrosinase and B16 melanoma cells. Most of the phenolic compounds significantly scavenged DPPH, superoxide, and hydroxyl radicals in a dose-dependent manner. Particularly, DFP showed potent DPPH (IC50 = 38.46 microM) and superoxide (IC50 = 291.62 microM) radical scavenging activities, while DCP exhibited the strongest hydroxyl radical scavenging activity (IC50 = 120.55 microM). CFP also exerted moderate DPPH, superoxide, and hydroxyl radical scavenging activities. Meanwhile, DCP (IC50 = 181.73 microM) showed potent tyrosinase inhibitory activity toward l-tyrosine as the substrate, whereas DFP (IC50 = 733.64 microM) significantly inhibited melanin synthesis in B16 melanoma cells. These current results indicate that these three polyamine conjugates from corn bran may be useful potential sources of natural antioxidants and skin-whitening agents.     

Inhibitory effect of red koji extracts on mushroom tyrosinase

Red koji has been recognized as a cholesterol-lowering diet supplement because of it contains fungi metabolites, monacolins, which reduce cholesterol synthesis by inhibiting HMG-CoA reductase. In this study, water extracts of red koji were loaded onto a C(18) cartridge, and the acetonitrile eluate was collected as test fraction. Red koji water extracts and its C(18) cartridge acetonitrile eluent had total phenols concentrations of 5.57 and 1.89 mg/g of red koji and condensed tannins concentrations of 2.71 and 1.20 mg/g of red koji, respectively. Both exhibited an antioxidant activity and an inhibitory activity to mushroom tyrosinase. The higher antioxidant activity of the red koji acetonitrile eluent was due to the existence of a high percentage of condensed tannins. The results from the kinetic study for inhibition of mushroom tyrosinase by red koji extracts showed that the compounds in the extracts competitively inhibited the oxidation of tyrosine catalyzed by mushroom tyrosinase with an ID(50) of 5.57 mg/mL.     

miR-29a在酉州乌羊不同体组织中表达特征及其在B16细胞中的动态表达分析

为探究miR-29a在酉州乌羊不同体组织表达特征及在B16细胞中的表达特性,本研究采用RT-qPCR方法检测miR-29a在酉州乌羊不同体组织以及B16细胞不同发育时期的表达水平。结果表明,miR-29a在酉州乌羊各体组织均有表达,其中在大脑中的相对表达量最高,肺和皮肤中的相对表达量最低;同时在渝东白羊皮肤中的相对表达量是酉州乌羊皮肤的3.44倍,且差异极显著(P<0.01);在细胞增殖过程中,miR-29a相对表达量极显著增加(P<0.01);在分化初期miR-29a相对表达量较高,之后趋于稳定且差异不显著(P>0.05)。在加入分化培养基后,细胞的形态发生变化,黑色素分泌增多。综上所述,miR-29a在酉州乌羊各组织中广泛表达,miR-29a的相对表达量随着B16细胞的增殖而递增,但随着B16细胞的分化而降低,推测miR-29a与B16细胞的增殖与分化密切相关。本研究结果为明确miR-29a在酉州乌山羊各组织中的表达特性及其是否参与了黑色素细胞行为学的调控提供了一定的理论证据。     

Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols

Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERbeta but not via ERalpha. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERbeta. HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species.     

Berry phenolic extracts modulate the expression of p21(WAF1) and Bax but not Bcl-2 in HT-29 colon cancer cells

Previous studies have shown that anthocyanin-rich berry extracts inhibit the growth of cancer cells in vitro. The objective of this study was to compare the effects of berry extracts containing different phenolic profiles on cell viability and expression of markers of cell proliferation and apoptosis in human colon cancer HT-29 cells. Berry extracts were prepared with methanol extraction, and contents of the main phenolic compounds were analyzed using HPLC. Anthocyanins were the predominant phenolic compounds in bilberry, black currant, and lingonberry extracts and ellagitannins in cloudberry extract, whereas both were present in raspberry and strawberry extracts. Cells were exposed to 0-60 mg/mL of extracts, and the cell growth inhibition was determined after 24 h. The degree of cell growth inhibition was as follows: bilberry > black currant > cloudberry > lingonberry > raspberry > strawberry. A 14-fold increase in the expression of p21WAF1, an inhibitor of cell proliferation and a member of the cyclin kinase inhibitors, was seen in cells exposed to cloudberry extract compared to other berry treatments (2.7-7-fold increase). The pro-apoptosis marker, Bax, was increased 1.3-fold only in cloudberry- and bilberry-treated cells, whereas the pro-survival marker, Bcl-2, was detected only in control cells. The results demonstrate that berry extracts inhibit cancer cell proliferation mainly via the p21WAF1 pathway. Cloudberry, despite its very low anthocyanin content, was a potent inhibitor of cell proliferation. Therefore, it is concluded that, in addition to anthocyanins, also other phenolic or nonphenolic phytochemicals are responsible for the antiproliferative activity of berries.     

Glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots

Tyrosinase is known to be a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair. Various dermatological disorders, such as melasama, age spots, and sites of actinic damage, arise from the accumulation of an excessive level of epidermal pigmentation. The inadequacy of current therapies to treat these conditions as well as high cytotoxicity and mutagenicity, poor skin penetration, and low stability of formulations led us to seek new whitening agents to meet the medical requirements for depigmenting agents. The inhibitory effect of licorice extract on tyrosinase activity was higher than that expected from the level of glabridin in the extract. This led us to test for other components that may contribute to this strong inhibitory activity. Results indicated that glabrene and isoliquiritigenin (2',4',4-trihydroxychalcone) in the licorice extract can inhibit both mono- and diphenolase tyrosinase activities. The IC(50) values for glabrene and isoliquiritigenin were 3.5 and 8.1 microM, respectively, when tyrosine was used as substrate. The effects of glabrene and isoliquiritigenin on tyrosinase activity were dose-dependent and correlated to their ability to inhibit melanin formation in melanocytes. This is the first study indicating that glabrene and isoliquiritigenin exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, suggesting that isoflavenes and chalcones may serve as candidates for skin-lightening agents.     

Evaluation of the estrogenic effects of legume extracts containing phytoestrogens

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.