Acetyl- and butyrylcholinesterase inhibitory activities of sterols and phlorotannins from Ecklonia stolonifera

ABSTRACT:   As part of this study on the isolation of cholinesterase inhibitors from natural marine products, the bioactivity of the ethanolic extracts from 27 Korean seaweeds were screened using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory assays. Ecklonia stolonifera exhibited promising inhibitory properties against both AChE and BChE. Bioassay-guided fractionation of the active n -hexane and ethyl acetate (EtOAc) soluble fractions, obtained from the ethanolic extract of E. stolonifera , resulted in the isolation of the sterols; fucosterol ( 1 ) and 24-hydroperoxy 24-vinylcholesterol ( 2 ), from the n -hexane fraction and the phlorotannins; phloroglucinol ( 3 ), eckstolonol ( 4 ), eckol ( 5 ), phlorofucofuroeckol-A ( 6 ), dieckol ( 7 ), triphlorethol-A ( 8 ), 2-phloroeckol ( 9 ) and 7-phloroeckol ( 10 ), from the EtOAc fraction. Of these, compounds 2 , 9 and 10 were isolated from E. stolonifera for the first time. Compounds 4 – 7 , 9 and 10 exhibited inhibitory potential against AChE, with 50% inhibition concentration (IC₅₀) values of 42.66 ± 8.48, 20.56 ± 5.61, 4.89 ± 2.28, 17.11 ± 3.24, 38.13 ± 4.95 and 21.11 ± 4.16 μM, respectively; whereas, compounds 1 , 2 , 4 and 6 were found to be active against BChE, with IC₅₀ values of 421.72 ± 1.43, 176.46 ± 2.51, 230.27 ± 3.52 and 136.71 ± 3.33 μM, respectively. It has been suggested that the inhibition of these enzymes by the sterols and phlorotannins derived from marine brown algae could be a useful approach for the treatment of Alzheimer's disease.     

水产品中血管紧张素转换酶抑制肽的研究进展

综述了国内外水产品中血管紧张素转换酶抑制剂的研究进展,总结了血管紧张素转换酶抑制剂的作用机理、制备工艺,对 ACEIP 的构效关系作了探讨,对水生生物资源中 ACEIP 的开发具有一定的指导意义。     

虾头内源酶酶解虾肉产物的ACE抑制活性及其降低原发性高血压老鼠血压的效果

虾头内源酶酶解虾肉产物的ACE抑制活性及其降低原发性高血压老鼠血压的效果=Angiotensin I converting enzyme inhibitory activity of shrimp meat hydrolysate by endogenous enzymes from shrimp head and its antihypertensive effects on spontaneously hypertensive rats［刊,中］/洪鹏志（广东海洋大学食品科技学院,湛江524088）,曹文红,郝更新,章超桦//水产学报,—2007,31(1).-76～83 在pH 7.35、温度57.2 ℃、水解时间4 h、虾肉/虾头为1∶1,底物浓度为20%（W/V）的条件下,利用虾头的内源酶酶解虾肉（南美白对虾）,制备了具有血管紧张素转化酶（ACE）抑制活性的酶解产物,其对ACE的抑制率为41.9%（2 mg·mL-1）。通过凝胶过滤层析测定出酶解产物的分子量分布较广,在7.4×104至29.7之间。分子量在2.7×103以下的凝胶层析组分占酶解产物氨基酸总量的83.1%。收集的高活性组分分子量在959～338之间,其IC50为0.32 mg·mL-1苯丙氨酸、疏水氨基酸（亮氨酸、异亮氨酸和缬氨酸）和芳香族氨基酸（脯氨酸、酪氨酸和色氨酸）占氨基酸总量的42.33%。体内试验的结果显示,南美白对虾的虾肉虾头内源酶酶解产物经灌喂原发性高血压老鼠后显示较强的降血压活性。 图4表2参13 关键词：南美白对虾;内源酶;血管紧张素转化酶抑制活性;抗高血压作用 E-mail:zhangch@gdou.edu.cn     

3942中性蛋白酶酶解中国毛虾蛋白制备血管紧张素转移酶抑制肽

采用3942中性蛋白酶酶解中国毛虾(Acetes chinensis)蛋白，制备具有抑制血管紧张素转移酶(ACE)活性的多肽。采取正交实验研究料水比、酶量、反应温度、反应时间4个因素对酶解中国毛虾蛋白产物的ACE抑制活性的影响。结果显示，在料水比1：3(体积比)，酶量0．5％，温度45℃，时间8h的水解条件下得到的酶解产物F7的ACE抑制活性最高，其抑制率达到92．86％。用层析柱Sephadex G-25、SP-Sephadex C-50对F7进行分离纯化，选取ACE抑制活性最高的峰进一步用反相高压液相色谱进行纯化，得到单一组分FⅠ。经飞行时间质谱和碰撞诱导裂解分析确定，FⅠ为新型的ACE抑制肽Ser-Pro，IC50值为272μmol／L。     

Identification of angiotensin I-converting enzyme inhibitory peptides derived from salmon muscle and their antihypertensive effect

ABSTRACT:   The salmon peptide digested from salmon muscle showed a strong inhibitory activity against the angiotensin I-converting enzyme (ACE). The antihypertensive effect of the salmon peptide on spontaneously hypertensive rats (SHR) was examined. After the single intravenous administration of the salmon peptide at a dose of 30 mg/kg body weight, the systolic blood pressure (SBP) was significantly reduced against the control. Further, a double-blind, placebo-controlled, parallel-group study determined the efficacy of the salmon peptide in mild hypertensive subjects. The SBP, after a 1.0 g of salmon peptide intake, was significantly reduced at 4 weeks after the intake, and 2 weeks after the intake finished, compared to the value before ingestion. Bioassay-guided separation of the salmon peptide, using a combination of column chromatographic techniques, led to the identification of 20 active di- and tri-peptides, including Ile-Val-Phe and Phe-Ile-Ala as two new ACE inhibitory tripeptides. Ile-Trp had the strongest ACE inhibitory activity (IC₅₀ = 1.2 μM) in vitro , and contributed 5.2% to the total ACE inhibitory activity. The salmon peptide and Ile-Trp showed a digestive resistance by in vitro assay, which mimicked the digestive organ, and had no affinity for factors related to blood pressure regulation, except for the ACE inhibitory activity.     

褐藻多酚的生物活性及提取技术研究进展

本文对不同褐藻中的褐藻多酚存在的形式进行了分析,综述了褐藻多酚抗菌、抗氧化、抗凝血、抗肿瘤以及化学防御等生物活性的研究进展,并比较了有机溶剂浸提、超声波辅助提取、微波辅助提取以及有机溶剂萃取纯化等方法的提取效果,旨在为褐藻多酚的进一步研究和利用提供参考依据.     

Characterization of Collagen Enzymatic Hydrolysates Derived from Lizardfish (Synodus fuscus) Scales

Acid soluble collagen was extracted from the scales of lizardfish and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. After 8 h of collagen hydrolysis, hydrolysates had an estimated degree of hydrolysis (DH) from 4 ± 0.05 to 25 ± 0.63% (p < 0.05). All hydrolysates had angiotensin converting enzyme inhibitory and antioxidant activity. These activity levels showed little change after treatment with gastrointestinal proteases. Results indicated that the lizardfish by-products may be improved by enzymatic treatment with acid-soluble collagen from lizardfish scales.     

中国毛虾ACE抑制肽的初步研究

对以中国毛虾为原料酶法制备具有抑制血管紧张素转换酶(ACE)活性的酶解产物的方法作了探讨。以体外活性(ACE抑制率)为指标,通过正交试验确定胃蛋白酶的最佳酶解条件为pH2.4、温度41℃、酶量900U·g-1底物、底物浓度8%;酶解产物的IC50为0.65mg·mL-1,再分别利用SephadexG 25和SephadexG 15对其进行进一步分离提纯,IC50降至0.084mg·mL-1和0.046mg·mL-1,活性组分中的疏水性氨基酸含量增高,最终活性产物的分子量分布在700～1900。     

血管紧张素转换酶抑制肽的分离

采用截留分子量分别为30 ku、10 ku、6 ku、3 ku的中空纤维超滤膜对扇贝酶解产物进行分级分离,并结合凝胶柱层析分离技术测定了各分离组分的分子量分布,同时还测定了各分离组分对血管紧张素转换酶(ACE)的抑制活性。实验结果表明,不同截留分子量超滤膜可以实现酶解产物按其分子量大小的分级分离,不同分离组分的ACE抑制力活性差异较大,总趋势是截留分子量相对较小的超滤膜透过液的ACE抑制活性较强。其中,截留分子量为3 ku超滤膜的透过液具有最强的ACE抑制活性,其ACE抑制率I(%)=96.17%,半抑制浓度IC_(50)= 0.078 mg·mL~(-1)。通过对截留分子量为3 ku超滤膜的透过液依次经过Sephadex G-25及Sephadex G-15凝胶柱层析分离纯化后,分离出了ACE抑制活性最强的2个组分活性肽,其平均分子量分别为1 300 u和900 u左右,其IC_(50)分别为0.026 mg·mL~(-1)和0.012 mg·mL~(-1)。     

鼠尾藻多酚分级组分的抑菌活性研究

采用平板生长抑制法对鼠尾藻Sargassum thunbergiikuntze多酚化合物各分级组分进行抑菌活性研究。结果表明,在一定浓度范围内各分级组分对副溶血弧菌、哈维氏弧菌、沙蚕弧菌、金黄色葡萄球菌、大肠杆菌、鳗弧菌、溶藻弧菌和四联微球菌等受试菌株均有抑菌活性。其中,分级组分I(Mr5.0×103)的抑菌效果优于其他组分,对副溶血弧菌、哈维氏弧菌和沙蚕弧菌的最低抑菌浓度(MIC)值为900μg/ml,对金黄色葡萄球菌、大肠杆菌、鳗弧菌和溶藻弧菌的MIC值为1 800μg/ml,对四联微球菌的MIC值为3 600μg/ml。另外,组分I(Mr5.0×103)显示出较好的热稳定性,在pH 3~4时对受试菌的抑菌活性最强。     

超低温保存对罗氏沼虾胚胎几种酶活性的影响

研究了超低温冷冻保存(-196℃)对罗氏沼虾胚胎内总三磷酸腺苷酶(ATPase)、肌酸激酶(CK)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)等酶活性的影响。运用试剂盒分别测定了冷冻前后罗氏沼虾胚胎内酶活性的变化。结果表明,经过超低温冷冻保存后,胚胎内7种酶的活性均显著下降(P0.05),其中总ATPase、CK、LDH和SDH的活性分别从冻前的1.322±0.162 U/mg prot、0.404±0.015 U/mg prot、352.225±23.214 U/gprot和2.067±0.139 U/mg prot下降到0.087±0.003 U/mg prot、0.010±0.002 U/mg prot、5.890±0.658 U/gprot和0.552±0.138 U/mg prot;SOD、CAT和GSH-Px的活性分别从冻前的19.217±0.677 U/mg prot、3.587±0.233 U/mg prot和7.626±1.106 U/(min.mg)下降至3.579±0.234 U/mg prot、1.773±0.227 U/mg prot和1.524±0.096 U/(min.mg);MDA的活性从1.015±0.038 n mol/mg prot上升到20.937±0.320 n mol/mgprot,超低温冷冻对罗氏沼虾胚胎酶活性有显著性影响。     

超低温冷冻对斑尾刺虾虎鱼卵中酶活性的影响

研究了超低温冷冻保存(-196℃)对斑尾刺虾虎鱼成熟卵中总ATP酶、肌酸激酶(CK)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)和丙二醛(MDA)等酶活性的影响。结果表明,经过冷冻保存后,总ATP酶、CK、SDH和LDH的活性显著下降(P<0.05),同时可见添加抗冻剂组比未添加抗冻剂组下降幅度更大(P<0.05);SOD、CAT和GSH-PX的活性也显著下降(P<0.05),未添加抗冻剂组比添加抗冻剂组下降更明显(P<0.05);MDA的活性显著升高,且未添加抗冻剂组显著高于添加抗冻剂组(P<0.05)。超低温冷冻和是否添加抗冻剂都对斑尾刺虾虎鱼卵中酶活性有显著性影响。     

超低温冷冻保存对大黄鱼精子酶活性的影响

研究超低温(-196℃)冷冻保存对大黄鱼(Pseudosiaena crocea)精子内总ATP酶、肌酸激酶(CK)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)等酶活性的影响。运用试剂盒分别测定了冷冻前后大黄鱼精子内酶活性的变化。结果表明,经过超低温冷冻保存后,大黄鱼精子的活力下降,精子内GR活性从(4.42±0.29)U·L-1增加到(58.93±2.26)U·L-1(P<0.05);其它几种酶的活性均显著下降(P<0.05),总ATP酶、CK、SDH的活性分别从冻前的(60.16±5.88)U·mL-1、(11.91±0.76)U·mL-1和(51±2.16)U·mL-1下降到(3.54±0.37)U·mL-1、(10.22±0.32)U·mL-1和(31.5±2.08)U·mL-1;LDH、SOD和CAT活性从冻前的(7 806.44±110.11)U·L-1、(42.65±1.56)U·mL-1和(119.91±8.10)U·mL-1下降到(2 654.13±70.06)U·L-1、(31.99±1.57)U·mL-1和(55.87±2.32)U·mL-1。超低温冷冻保存对大黄鱼精子活力和精子酶活性均有显著影响。     

Potential Precursor of Angiotensin-I Converting Enzyme (ACE) Inhibitory Activity and Structural Properties of Peptide from Peptic Hydrolysate of Cutlassfish Muscle

ABSTRACT

Peptic hydrolysates were prepared by digesting the cutlassfish muscle protein using pepsin for 1, 3, and 6 h, and their inhibitory activity against angiotensin-I converting enzyme (ACE) was studied. The ACE-inhibitory effect of the peptic hydrolysate of cutlassfish muscle generated at the 3 h time point exhibited the strongest activity. After identifying the optimal hydrolysate, the active peptide was isolated by ultrafiltration, gel permeation, and high performance liquid chromatography (HPLC). The resulting purified peptide was characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and was identified to be a 496.44 Da pentapeptide (Phe-Ser-Gly-Gly-Glu). The ACE-inhibitory activity of the active peptide exhibited an IC₅₀ value of 0.033 ± 0.003 mg/ml. A molecular docking program was used to simulate the interaction between the peptide and ACE, which revealed that the inhibitory effect was mainly due to the hydrogen bonds between ACE and the peptide. Based on the ACE-inhibitory properties and the molecular docking study of the resulting active peptide, we demonstrated an increase in nitric oxide (NO) production in a dose-dependent manner. In conclusion, cutlassfish protein hydrolysate and the resulting active peptide could be used as active ingredients in functional food as anti-hypertensive agents.     

盐度对点篮子鱼消化酶活性的影响

以自然海水组(盐度30～32)为对照,研究了20、10、5三种盐度对点篮子鱼(平均体重86.09 4-17.99g,平均全长13.90±1.53 cm)幽门盲囊、肠、胃和肝脏中蛋白酶、淀粉酶和脂肪酶活力的影响.结果表明:第24天盐度20的点篮子鱼幽门盲囊蛋白酶活力显著低于对照组(P<0.05);第24天和第48天,在同...     

中国对虾亲虾生殖周期中消化酶活性及组织生化成分的变化

叙述了中国对虾亲虾(海捕亲虾与池养亲虾)在生殖周期中肝胰腺、中肠的消化酶活性及卵巢、肝胰腺、血淋巴生化成分的研究结果。蛋白酶、脂肪酶在卵巢成熟的Ⅳ期前活性较高，临产卵时(Ⅴ期)活性明显下降，而且海捕亲虾与池养亲虾具有相同的变化趋势；中国对虾卵巢、肝胰腺和血淋巴的蛋白质和脂肪含量随性腺发育有不同的变化规律，其中总蛋自在这几种组织中均随性腺发育含量增加，至Ⅳ期(肝胰腺)和Ⅴ期(卵巢、血淋巴)达到高峰值；卵巢的总脂和甘油三酯从发育Ⅰ期-Ⅴ期随着性腺指数的增长而有规律的上升，但肝胰腺脂肪含量在卵巢发育初期(Ⅰ期-Ⅱ期)未见明显变化，Ⅱ期以后，肝胰腺脂肪含量逐渐下降，当卵巢脂肪含量达到最高值时，肝胰腺脂肪含量正好下降到最低点。同时，还对中国对虾卵巢发育过程中蛋白质和脂肪的积累问题进行了分析和讨论。     

大口黑鲈肝细胞中恩诺沙星的消除及其代谢酶活性的测定

建立了反相高效液相色谱法检测大口黑鲈(Micropterus salmoides)原代肝细胞中恩诺沙星(Enrofloxacin,EF)代谢情况的方法,并通过其代谢产物环丙沙星(Ciprofloxacin,CF)来间接反映恩诺沙星N-脱乙基酶的活性。采用二氯甲烷提取细胞中的药物,正己烷去脂,反相高效液相色谱法测定其中的药物浓度。经测得该方法样品回收率均大于80.28%,日间变异系数小于5.54%。通过EF与大口黑鲈肝细胞进行孵育,发现EF在细胞中的消除方程为C=50e-0.0008t,消除半衰期(T1/2)为86.63 h,消除速率常数(K)为8×10-4/h。对孵育过程中的酶活研究发现最佳孵育时间为45 min,其酶活性为0.25μg/(min.mg),酶动力学参数中最大反应速率(Vmax)为0.29μg/(min.mg),米氏常数(Km)为22.37μg/mL,内在清除率(Clint)为0.01 mL/(min.mg)。结果表明,该酶与底物结合力不强,酶促反应强度较弱,EF在大口黑鲈肝细胞中的代谢较缓慢。     

血管紧张素转化酶抑制肽研究进展

为研究鳗鱼加工副产物的综合利用,以鳗鱼骨胶原蛋白为原料,采用酶解法制备血管紧张素转化酶 (Angiotensin-converting enzyme, ACE) 抑制肽。以ACE抑制活性和水解度为评价指标,通过单因素和响应面试验确定最佳的酶解制备条件,并分析酶解产物的分子质量分布和氨基酸组成。结果显示,碱性蛋白酶为最适水解酶,最佳酶解条件为：温度50 ℃,质量浓度15 g·L^–1,酶解时间5.25 h,加酶量3.1% (质量分数),pH 9.2。在此条件下,鳗鱼胶原蛋白肽的ACE抑制活性为70.33%,与预测值接近;酶解产物中分子质量小于1 kD的肽占57.02%,1~3 kD的肽占36.55%;氨基酸组成分析表明,酶解产物中与ACE抑制活性有关的疏水性氨基酸 (如脯氨酸、缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸) 含量增加。     

超低温冷冻对脊尾白虾精子几种酶活性的影响

研究了超低温冷冻保存(-196℃)对脊尾白虾精子内琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、Na+/K+-ATP酶、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)与顶体酶活性的影响,以期为提高脊尾白虾精子超低温冷冻效果提供理论依据.设置对照组(未添加抗冻剂)、3个实验组[分别添加DMSO (V/V) 10.0％、12.5％、15.0％],于冷冻0、1、3、5、7、15d取样测定各酶活性.结果表明,经冷冻后,除GR外,其他所测酶活性均出现显著下降(P＜0.05),且以对照组酶活性下降幅度最大.GR活性在冷冻7d内显著升高,且对照组明显高于实验组(P＜0.05),在冷冻15d时又出现下降.添加15.0％ DMSO组所测酶活性均高于同期其它各组,表明15.0％ DMSO对精子内酶的保护作用较好.冷冻15 d后15.0％ DMSO组的SDH、LDH和Na+/K+-ATP酶活性由(28.500±1.453) U/mL、(1290.836±27.603) U/L和(2.605-0.232) μmol/(mg·h)分别降至(15.300±0.950) U/mL、(363.713-13.943) U/L和(0.542-0.186) μmol/(mg.h);SOD和CAT活性由(106.497±7.217) U/mL、(383.632±4.731)U/g分别降至(17.036 ±0.321)U/mL、(166.940±1.910) U/g;顶体酶活性从(3.521±0.010)μIU/106降至(1.212±0.043)μIU/106;而GR活性由(217.042±6.962) U/L上升至(302.787±24.558)U/L.从冷冻后各酶下降幅度来看,超低温冷冻对SOD活性的影响最大,其次是Na+/K+-ATP酶.