Physicochemical Characterization of Fucoidans from Sargassum henslowianum C.Agardh and Their Antithrombotic Activity In Vitro

Sargassum fucoidan is a kind of sulfated heteropolysaccharide with a variety of biological activities. The aim of this study was to investigate the extraction, purification, physicochemical characterization and in vitro antithrombotic activity of fucoidan from Sargassum henslowianum C.Agardh. Hot-water-assisted ultrasound was used to extract fucoidan (F). Fucoidan was purified by DEAE cellulose 52 (F1), Vc-H₂O₂ (FD1) and Superdex 75 gel (FDS1). The physical and chemical properties of fucoidans were analyzed by chemical composition, monosaccharide composition, average molecular weight (Mw) and FTIR. The sulfate contents of F, F1, FD1 and FDS1 were 11.45%, 16.35% and 17.52%, 9.66%, respectively; the Mw was 5.677 × 10⁵, 4.393 × 10⁵, 2.176 × 10⁴ and 6.166 × 10³, respectively. The results of monosaccharide composition showed that the four fucoidans contained l-fucose, d-galactose, l-mannose, d-xylose, l-rhamnose and d-glucose, but the mass fraction ratio was different. The results of FTIR showed that fucoidan contained characteristic peaks of sugar and sulfate. In vitro, F1, FD1 and FDS1 could alleviate HUVEC damage induced by adrenaline (Adr). F1, FD1 and FDS1 decreased vWF and TF and increased the ratio of t-PA/PAI-1 in Adr-induced HUVEC.     

Sulfated Polysaccharide Extracted from the Green Algae Codium bernabei: Physicochemical Characterization and Antioxidant,Anticoagulant and Antitumor Activity

Codium bernabei is a green alga that grows on Chilean coasts. The composition of its structural polysaccharides is still unknown. Hence, the aim of this work is to isolate and characterize the hot water extracted polysaccharide fractions. For this purpose, the water extracts were further precipitated in alcohol (TPs) and acid media (APs), respectively. Both fractions were characterized using different physicochemical techniques such as GC-MS, GPC, FTIR, TGA, and SEM. It is confirmed that the extracted fractions are mainly made of sulfated galactan unit, with a degree of sulfation of 19.3% (TPs) and 17.4% (ATs) and a protein content of 3.5% in APs and 15.6% in TPs. Other neutral sugars such as xylose, glucose, galactose, fucose, mannose, and arabinose were found in a molar ratio (0.05:0.6:1.0:0.02:0.14:0.11) for TPs and (0.05:0.31:1.0:0.03:0.1:0.13) for ATs. The molecular weight of the polysaccharide samples was lower than 20 kDa. Both polysaccharides were thermally stable (Tonset > 190 °C) and showed antioxidant activity according to the ABTS^•+ and DPPH tests, where TPs fractions had higher scavenging activity (35%) compared to the APs fractions. The PT and APTTS assays were used to measure the anticoagulant activity of the polysaccharide fractions. In general, the PT activity of the TPs and APs was not different from normal plasma values. The exception was the TPs treatment at 1000 µg mL⁻¹ concentration. The APTTS test revealed that clotting time for both polysaccharides was prolonged regarding normal values at 1000 µg mL⁻¹. Finally, the antitumor test in colorectal carcinoma (HTC-116) cell line, breast cancer (MCF-7) and human leukemia (HL-60) cell lines showed the cytotoxic effect of TPs and APs. Those results suggest the potential biotechnological application of sulfate galactan polysaccharides isolated from a Chilean marine resource.     

木麻黄小枝提取物的抗蚁及抑菌生物活性

采用滤纸药膜法测定木麻黄（Casuarina equistiolia Forst小枝提取液对黑翅土白蚁的毒杀和驱避活性,并测定了木麻黄小枝提取液对大肠杆菌、金黄色葡萄球菌、清酒假丝酵母、黑曲霉的抑菌活性。结果表明∶木麻黄小枝水提取液对黑翅土白蚁有显著的触杀毒性,处理浓度为5mg血L条件下,校正死亡率达82%。木麻黄小枝水提取液对黑翅土白蚁无显著的忌避活性,对金黄色葡萄球菌表现出显著的抑制活性。化学成分预试验表明,木麻黄小枝水提取液中含有黄酮类、酚类和糖类等代谢产物。     

Antioxidative and Antigenotoxic Activity of Extracts from Cosmos (Cosmos bipinnatus) Flowers

The Cosmos bipinnatus has been used in a traditional herbal remedy for various diseases such as jaundice, intermittent fever, and splenomegaly. The present study describes the preliminary evaluation of antioxidant activities and antigenotoxic effect of Cosmos bipinnatus flowers according to four different colors (white, pink, orange, and violet). The antioxidants properties were evaluated by determining TPC, DPPH RSA, ABTS RSA, and RP. The highest TPC of methanolic CFE (at concentration of 1 mg/ml) showed in violet colored CF (1,013 μM), and IC₅₀ of DPPH RSA, ABTS RSA, and RP were also the lowest in violet colored CFE with values of 0.61, 1.48, and 0.82 mg/ml, respectively. The antigenotoxic effect of the CFE on DNA damage induced by H₂O₂ in human leukocytes was evaluated by Comet assay. Pretreatments with CFE produced significant reductions in oxidative DNA damage at the concentration of 500 μg/ml, except for violet colored CFE. The ED₅₀ value of white colored CFE has shown the highest inhibition (0.40 mg/ml) on H₂O₂ induced DNA damage, followed by orange > pink > violet color. These results suggested that Cosmos bipinnatus has significant antioxidant activity and protective effect against oxidative DNA damage.     

糖胶树花次生代谢产物及其抗细菌活性

采用水蒸气蒸馏法提取糖胶树花挥发油,通过气相色谱-质谱联用技术(GC-MS)对挥发油进行成分分析,采用薄层层析——生物自显影法测定糖胶树花乙酸乙酯层提取物对供试细菌的抑制活性。结果表明,糖胶树花挥发油得率为0.018%,通过GC-MS分析,从中鉴定出20个成分,占挥发油总量的97.53%,其主要成分为2-莰烯(23.30%),芳樟醇(23.09%),(-)-4-萜品醇(10.54%)。糖胶树花乙酸乙酯层提取物对大肠杆菌表现出较强的抑制活性,其次为黄瓜角斑病菌,对其他供试细菌未表现出抑制活性。     

New Insights of Turmeric Extract-Loaded PLGA Nanoparticles: Development,Characterization and In Vitro Evaluation of Antioxidant Activity

Here, we presented new insights of the development of poly(lactic-co-glycolic acid) nanoparticles containing turmeric compounds (turmeric-PLGA-NPs) using emulsion-solvent evaporation method. The nanoparticulate system was characterized by size, zeta potential, morphology, release profile, partition parameter, stability and encapsulation efficiency (%EE). Antioxidant activity studies were also evaluated. The Korsmeyer-Peppas model (M_t/M_∞ vs. t) was used to determine the release mechanisms of the studied system. Our results demonstrated the emulsion-solvent evaporation method was shown advantageous for producing turmeric-PLGA-NPs in the range of 145 nm with high homogeneity in size distribution, zeta potential of ?21.8 mV and %EE about 72%. Nanoparticles were stable over a period of one month. In vitro study showed a release of curcumin governed by diffusion and relaxation of the polymeric matrix. The partition parameter of the extract in relation to blank-PLGA-NPs was 0.111?±?0.008 M^?1, indicating a low affinity of curcumin for the polymer matrix. Antioxidant ability of the turmeric-PLGA-NPs in scavenging the radical 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) was inferior to free turmeric extract and showed a concentration and time-dependent profile. The study concluded that PLGA nanoparticles are potential carriers for turmeric extract delivery.

     

大豆豆渣粗提物清除DPPH自由基活性及其协同效应的研究

用二苯代苦味肼基自由基(DPPH)-TLC法和酶标仪法对大豆豆渣石油醚、乙酸乙酯、丙酮、乙醇粗提物的自由基清除活性进行了定性和定量分析,考察了抗氧化剂维生素C、柠檬酸对大豆豆渣乙醇粗提物清除DPPH自由基活性的协同效应。结果表明:大豆豆渣不同极性粗提物均有一定的自由基清除活性,其中乙醇粗提物的自由基清除活性最强,在浓度为10.0 mg.mL-1,于37℃下保温15 min时,对0.4 mg.mL-1的DPPH自由基的清除率可达76.48%;维生素C和柠檬酸对大豆豆渣乙醇粗提物均能产生一定的协同效应,且维生素C的协同作用强于柠檬酸。     

Antioxidant and Antihypertensive Activity of Extract from Thymus serpyllum L. in Experimental Hypertension

The low incidence of cardiovascular disease in Mediterranean countries leads to an increased interest of the scientific community for the Mediterranean diet. Our aim was to evaluate total phenol and flavonoid contents, antioxidant capacity, free radical scavenging activity and potential antihypertensive effect of aqueous extract obtained from Thymus serpyllum L. (wild thyme, TE), an aromatic herb from the Lamiaceae family (highly present in Mediterranean diet), in spontaneously hypertensive rats (SHR) and in normotensive Wistar rats. Total phenol content of TE was 2008.33?±?10.6 mg/L GAE, and rosmarinic and caffeic acids were predominant phenolic compounds. The ferric reducing/antioxidant power and antioxidant capacity analysis revealed strong antioxidative properties of TE. In vitro nitric oxide-scavenging activity of 1 mg/l TE was 63.43 % with the IC₅₀ value of 122.36 μg/ml. Bolus injection of TE (100 mg/kg body weight i.v.) induced significant decrease of systolic and diastolic blood pressure and total peripheral resistance in SHR, without effects on these parameters in normotensive Wistar rats. Cardiac index remained unchanged after TE treatment in all experimental rats. Given dose of TE did not show significant nitric oxide-scavenging activity in vivo. Our results indicate that TE may protect against hypertension in experimental model of essential hypertension.     

大叶桃花心木的花、叶抽提物对斜纹夜蛾的拒食活性

以大叶桃花心木的花和叶为材料,测试了其乙醇抽提物对重要多食性害虫斜纹夜蛾5龄幼虫的拒食活性。结果表明:试剂中大叶桃花心木的花与叶各自的抽提物质量分数5％时均有较强的拒食作用,非选择性拒食率分别高达96.33％和86.23％,选择性拒食率分别高达96.63％和91.98％;质量分数5％大叶桃花心木花、叶抽提物拒食率在48 h后均有所下降,但仍然保持较高的拒食率,其非选择性拒食率分别为87.3％和73.9％,选择性拒食率分别为95.46％和85.63％;连续以质量分数1％的花、叶抽提物浸叶饲喂时,试虫体重的增长明显低于对照试虫,处理后第5天,质量分数1％的花、叶抽提物处理试虫的平均体重分别为对照的49.32％和53.01％。研究结果表明,大叶桃花心木的花、叶的乙醇抽提物对斜纹夜蛾均具有良好的拒食活性,并且大叶桃花心木的花抽提物对斜纹夜蛾的拒食活性高于其叶的抽提物。      

Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL⁻¹, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC₅₀ of 2.41 µg·mL⁻¹. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.     

大叶冬青苦丁茶多糖提取、纯化与抗氧化活性研究

大叶冬青苦丁茶经85%乙醇溶液脱脂、热水提取、S-8大孔树脂脱色、醇沉、干燥,得到大叶冬青苦丁茶粗多糖。粗ILPS经DEAE-52纤维素阴离子交换层析柱分离,得到4个多糖组分ILPS-1,ILPS-2,ILPS-3和ILPS-4。采用化学法分别测定了粗多糖及其纯化组分的体外清除自由基（DPPH.自由基,O2^-.自由基,.OH自由基）能力、还原能力以及螯合金属离子能力。结果表明,大叶冬青苦丁茶多糖具有较强的体外抗氧化活性,并且抗氧化能力与多糖浓度之间存在良好的相关性。     

Polyoxygenated Steroids from the Octocoral Leptogorgia punicea and in Vitro Evaluation of Their Cytotoxic Activity

Five new polyoxygenated marine steroids—punicinols A–E (1–5)—were isolated from the gorgonian Leptogorgia punicea and characterized by spectroscopic methods (IR, MS, ¹H, ¹³C and 2-D NMR). The five compounds induced in vitro cytotoxic effects against lung cancer A549 cells, while punicinols A and B were the most active, with IC₅₀ values of 9.7 μM and 9.6 μM, respectively. The synergistic effects of these compounds with paclitaxel, as well as their effects on cell cycle distribution and their performance in the clonogenic assay, were also evaluated. Both compounds demonstrated significant synergistic effects with paclitaxel.     

茶树花Cu/Zn-SOD酶活性及其基因表达分析

为研究Cu/Zn-SOD基因在茶树花不同发育阶段、不同品种间的表达规律以及Cu/Zn-SOD基因表达量与酶活性之间的关系,本研究将‘龙井43’品种茶树花分为4个发育阶段,选择15个品种的茶树花为研究对象,采用实时荧光定量PCR技术对3个Cu/Zn-SOD基因表达水平进行定量分析,同时测定Cu/Zn-SOD酶活性。结果表明:在茶树花的4个发育阶段中,第4阶段Cu/Zn-SOD酶活性最低,与其他3个阶段具有显著差异;不同品种的茶树花Cu/Zn-SOD酶活性有较大差异,其中‘紫鹃’茶树花Cu/Zn-SOD酶活性最高,其次是‘黄金芽’和‘白叶1号’,‘黄观音’的Cu/Zn-SOD酶活性最低。茶树花不同发育阶段中Cu/Zn-SOD基因表达水平存在较大差异,细胞质型Cu/Zn-SOD1基因表达水平远高于叶绿体型Cu/Zn-SOD2和过氧化物酶体Cu/Zn-SOD3;不同品种茶树花Cu/Zn-SOD基因表达水平有较大差异,Cu/Zn-SOD1基因在‘黄牡丹’茶树花中表达量最高,Cu/Zn-SOD2基因在‘白叶1号’和‘黄观音’茶树花中表达量最高,Cu/Zn-SOD3基因在‘紫鹃’茶树花中的表达量最高。相关性分析得知‘龙井43’茶树花不同发育阶段Cu/Zn-SOD酶活性与Cu/Zn-SOD基因表达量之间的相关性不明显,而不同品种茶树花的Cu/Zn-SOD酶活性与Cu/Zn-SOD3基因表达量存在显著相关性。本研究初步明确了Cu/Zn-SOD酶活性较高的茶树品种和茶树花适宜的采摘时间,为茶树花SOD进一步研究提供参考。     

Production,Characterization and Immunomodulatory Activity of an Extracellular Polysaccharide from Rhodotorula mucilaginosa YL-1 Isolated from Sea Salt Field

A novel exopolysaccharide from marine-derived red yeast Rhodotorula mucilaginosa strain YL-1 was produced and characterized. The highest yield of polysaccharide reached 15.1 g/L after medium and culture parameter optimization. This exopolysaccharide, composed of four neural monosaccharides including glucose, mannose, galactose and fucose, had an average molecular weight of 1200 KDa. It had good immunomodulatory activity on RAW256.7 cell lines. ELISA (enzyme linked immunosorbent assay) and Q-PCR (quantitative real-time PCR) results showed that the cell was stimulated to express more IL-6, IL-18, IL-1β and TNFα cytokines than the control group. This is the first report of an exopolysaccharide with immunomodulatory activity from marine-derived Rhodotorula mucilaginosa.     

Aphicidal Activity of an Ageraphorone Extract From Eupatorium adenophorum Against Pseudoregma bambucicola (Homoptera: Aphididae,Takahashi)

The bamboo aphid, Pseudoregma bambucicola, is an important insect pest of bamboo that affects normal bamboo growth and induces sooty molds. The control of P. bambucicola involves the application of chemicals, such as imidacloprid, to which many species are resistant. In this study, we isolate a novel botanical pesticide (9-oxo-10,11-dehydro-ageraphorone) from an Eupatorium adenophorum(Asteraceae: Compositae) petroleum ether extract and test the aphicidal activity of this compound against P. bambucicola in laboratory bioassay and field-based experiments. This ageraphorone compound at a concentration of 2 mg/ml caused 73.33% mortality (corrected mortality [Subtracted the mortality of the negative control]: 70%) of P. bambucicola by laboratory bioassay within 6 h. Even at lower concentrations, this compound caused greater 33% mortality (corrected mortality: 30%) of aphids. Field experiments with naturally infested bamboo plants showed that two applications of 2 mg/ml ageraphorone to infested plants completely cleared infestations within 30 d. These effects were similar to those of the positive control (imidacloprid). These results reveal that 9-oxo-10,11-dehydro-ageraphorone exhibits significant aphicidal activity against bamboo aphids. We suggest that future research be directed at developing this ageraphorone compound from E. adenophorum as an aphicidal agent for biocontrol.     

Production and Physicochemical Characterization of Gelatin and Collagen Hydrolysates from Turbot Skin Waste Generated by Aquaculture Activities

Rising trends in fish filleting are increasing the amount of processing by-products, such as skins of turbot, a flatfish of high commercial value. In line with circular economy principles, we propose the valorization of turbot skins through a two-step process: initial gelatin extraction described for the first time in turbot, followed by hydrolysis of the remaining solids to produce collagen hydrolysates. We assayed several methods for gelatin extraction, finding differences in gelatin properties depending on chemical treatment and temperature. Of all methods, the application of NaOH, sulfuric, and citric acids at 22 °C results in the highest gel strength (177 g), storage and loss moduli, and gel stability. We found no relation between mechanical properties and content of pyrrolidine amino acids, but the best performing gelatin displays higher structural integrity, with less than 30% of the material below 100 kDa. Collagen hydrolysis was more efficient with papain than alcalase, leading to a greater reduction in Mw of the hydrolysates, which contain a higher proportion of essential amino acids than gelatin and show high in vitro anti-hypertensive activity. These results highlight the suitability of turbot skin by-products as a source of gelatin and the potential of collagen hydrolysates as a functional food and feed ingredient.     

Purification,Characterization and in vitro Anti-Tumor Activity of Proteins from Arca subcrenata Lischke

Two purified proteins G-6 and G-4-2 were obtained from Arca subcrenata Lischke using the homogenization, salting-out with ammonium sulfate, ion-exchange chromatography and gel filtration chromatography techniques. The purity of G-6 and G-4-2 was over 96%, as measured by RP-HPLC. G-6 and G-4-2 were measured by SDS-PAGE and IEF-PAGE to have molecular weights of 8.2 kDa and 16.0 kDa, and isoelectric points of 6.6 and 6.1, respectively. The amino acid constituents of G-6 and G-4-2 were also determined. The existence of saccharides in G-6 was demonstrated by the phenol-sulfuric acid method. G-6 and G-4-2 inhibited the proliferation of human tumor cells in vitro. By MTT assay, the IC₅₀ values of G-4-2 were 22.9 μg/mL, 46.1 μg/mL and 57.7 μg/mL against Hela, HL-60 and KB cell lines, respectively, and the IC₅₀ value of G-6 against HL-60 cell line was measured to be 123.2 μg/mL.     

Structural Characterization and Heparanase Inhibitory Activity of Fucosylated Glycosaminoglycan from Holothuria floridana

Unique fucosylated glycosaminoglycans (FG) have attracted increasing attention for various bioactivities. However, the precise structures of FGs usually vary in a species-specific manner. In this study, HfFG was isolated from Holothuria floridana and purified by anion exchange chromatography with the yield of ~0.9%. HfFG was composed of GlcA, GalNAc and Fuc, its molecular weight was 47.3 kDa, and the -OSO₃⁻/-COO⁻ molar ratio was 3.756. HfFG was depolymerized by a partial deacetylation–deaminative cleavage method to obtain the low-molecular-weight HfFG (dHfFG). Three oligosaccharide fragments (Fr-1, Fr-2, Fr-3) with different molecular weights were isolated from the dHfFG, and their structures were revealed by 1D and 2D NMR spectroscopy. HfFG should be composed of repeating trisaccharide units -{(L-FucS-α1,3-)d-GlcA-β1,3-d-GalNAc_4S6S-β1,4-}-, in which sulfated fucose (FucS) includes Fuc_2S4S, Fuc_3S4S and Fuc_4S residues linked to O-3 of GlcA in a ratio of 45:35:20. Furthermore, the heparanase inhibitory activities of native HfFG and oligosaccharide fragments (Fr-1, Fr-2, Fr-3) were evaluated. The native HfFG and its oligosaccharides exhibited heparanase inhibitory activities, and the activities increased with the increase of molecular weight. Additionally, structural characteristics such as sulfation patterns, the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibiting activity.     

茶叶提取物对B16小鼠黑色素瘤细胞的生物学效应及机理研究

研究了茶叶提取物对体外培养的B16小鼠黑色素瘤细胞的生物学效应,并对其机理进行初步探讨。分别用茶黄素单体（TF1）、EGCG、纯度为40%的茶黄素（TF40）处理细胞,然后观察细胞形态,并以溴化二苯四偶氮法（MTT法）测定受试物对黑色素细胞活力的影响,以左旋多巴为底物测定细胞内酪氨酸酶活性,采用比色法测定细胞中的黑色素含量。实验结果表明,各化合物对B16黑色素瘤细胞形态有不同程度的影响,对细胞活性、酪氨酸酶浓度和黑色素合成量有浓度依赖性抑制作用。这3种茶提取物能通过多种途径抑制黑色素合成,从而达到美白的效果,且茶叶提取物的效果优于Vc。