Blood glucose-lowering activity of protocatechuic acid is mediated by inhibiting a-glucosidase

α-Glucosidase inhibitors are effective in controlling postprandial hyperglycemia,which play crucial roles in the management of type 2 diabetes.Protocatechuic acid(PCA)is one of phenolic acids existing not only in various plant foods but also as a major microbial metabolite of dietary anthocyanins in the large colon.The present study investigated the inhibitory mechanism of PCA on a-glucosidase in vitro and examined its effect on postprandial blood glucose levels in vivo.Results from in vitro experiments demonstrated that PCA was a mix-type inhibitor of a-glucosidase.Driven by hydrogen bonds and van der Waals interactions,PCA reversibly bound withα-glucosidase to form a stable a-glucosidase-PCA complex in a spontaneous manner.The computational simulation found that PCA could insert into the active cavity of a-glucosidase and establish hydrogen bonds with catalytic amino acid residues.PCA binding aroused the steric hindrance for substrates to enter active sites and caused the structural changes of interacted catalytic amino acid residues.PCA also exhibited postprandial hypoglycemic capacity in diabetic mice.This study may provide the theoretical basis for the application of PCA as an active ingredient of functional foods in dietary management of diabetes.

Structural characterizations andα-glucosidase inhibitory activities of four Lepidium meyenii polysaccharides with different molecular weights

Four polysaccharides(MCPa,MCPb,MCPc,MCPd)were obtained from Lepidium meyenii Walp.Their structures were characterized by chemical and instrumental methods including total sugar,uronic acid and protein content determi-nation,UV,IR and NMR spectroscopy,as well as monosaccharide composition determination and methylation analy-ses.Four polysaccharides were a group of glucans with different molecular weights ranging from 3.12 to 14.4 kDa,and shared a similar backbone chain consisting of(1→4)-glucose linkages with branches attached to C-3 and C-6.Furthermore,bioactivity assay showed that MCPs had concentration-dependent inhibitory activity onα-glucosidase.MCPb(Mw=10.1 kDa)and MCPc(Mw=5.62 kDa)with moderate molecular weights exhibited higher inhibitory activ-ity compared with MCPa and MCPd.

Characterization of Angiotensin-? Converting Enzyme Inhibiting Peptide from Venerupis philippinarum with Nano-Liquid Chromatography in Combination with Orbitrap Mass Spectrum Detection and Molecular Docking

The complexity and diversity of peptide mixture from protein hydrolysates make their characterization difficult. In this study, a method combining nano LC-MS/MS with molecular docking was applied to identifying and characterizing a peptide with angiotensin-? converting enzyme(ACE-I) inhibiting activity from Venerupis philippinarum hydrolysate. Firstly, ethanol supernatant of V. philippinarum hydrolysate was separated into active fractions with chromatographic methods such as ion-exchange chromatography and high performance liquid chromatography in combination. Then seven peptides from active fraction were identified according to the searching result of the MS/MS spectra against protein databases. Peptides were synthesized and subjected to ACE-Iinhibition assay. The peptide NTLTLIDTGIGMTK showed the highest potency with an IC_(50) of 5.75 μmol L^(-1). The molecular docking analysis showed that the ACE-I inhibiting peptide NTLTLIDTGIGMTK bond with residues Glu123, Glu403, Arg522, Glu376, Gln281 and Asn285 of ACE-I. Therefore, active peptides could be identified with the present method rather than the traditional purification and identification strategies. It may also be feasible to identify other food-derived peptides which target other enzymes and receptors with the method developed in this study.

New flavanone-monoterpene hybrids as α-glucosidase inhibitors from the root bark of Morus alba

Objective The Morus alba root bark is a well-known Chinese herbal medicine called Sang-Bai-Pi and has often been used to relieve the hyperglycemic symptom of diabetes patients.The current work aims to further explore its bioactive constituents with α-glucosidase inhibitory activity for the potential treatment of diabetes.Methods A combination of different separating techniques including routine column chromatograph and HPLC especially on chiral columns were applied for the isolation of target molecules,while comprehensive spectroscopic experiments comprising MS,NMR,ECD,etc.were carried out to complete the structural assignment.The anti-hyperglycemic property of the isolates was evaluated by an in vitro α-glucosidase inhibitory bioassay.Results Two pairs of new flavanone-monoterpene hybrid enantiomers were isolated and identified,and an interesting phenomenon of mutual transformation between these cometabolites were detected,which resulted in their regio-isomerization and enantiomerization.The bioassay results revealed remarkable α-glucosidase inhibitory activity for these fascinating molecules.Conclusions The Morus alba root bark is a rich source of bioactive flavonoid derivatives and deserves further investigations to develop new potential chemotherapies for diabetes control and treatment.

A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis

AIM: To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-κB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-κB p50 subunit.METHODS: Pancreatitis was induced in male Wistar rats by administering 2×100 μg/kg body weight of cholecystokininoctapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK.RESULTS: All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity,pancreatic levels of TNF-α and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-κB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide.According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis.CONCLUSION: Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-κB might be clinically useful for the suppression of the severity of acute pancreatitis.

ACE-I inhibitory peptide fractions from enzymatic hydrolysates of velvet bean (<i>Mucuna pruriens</i>)

The hydrolysis of velvet bean (Mucuna pruriens) protein in the presence of Alcalase?-Flavourzyme? and Pepsin-Pancreatin was investigated. The results showed that Alcalase?-Flavourzyme? (29.08%) sequential system catalyzed the hydrolysis most efficiently that Pepsin-Pancreatin (24.78%). In addition, the higher ACE-I inhibitory activity was achieved with the sequential system Alcalase?-Flavourzyme? (33.13%). Furthermore, the concentration of peptides employing an ultrafiltration (UF) system or their purification by gel filtration chromatography showed that the oligomeric peptides with lower molecular weight registered the highest ACE-I inhibitory activity. It has been demonstrated that Mucuna pruriens protein hydrolysates could serve as a source of peptides with ACE inhibitory activity and this activity can be attributed mainly to the mixture of short peptides in the hydrolysate.

白果肽的二肽基肽酶-Ⅳ抑制活性及其抑制机理研究

食源性二肽基肽酶-Ⅳ(dipeptidyl peptidaseⅣ,DPP-Ⅳ)抑制肽可有效调节机体内糖代谢,降低机体血糖水平。为评估白果肽(phenylalanine-alanine-proline-serine-tryptophan,FAPSW和methionine-proline-glycine-proline-proline,MPGPP)的降糖潜能,采用体外试验测定其DPP-Ⅳ抑制活性,并进一步通过分子对接和分子动力学模拟揭示其抑制机理。结果表明,FAPSW和MPGPP具有良好的DPP-Ⅳ抑制活性,且MPGPP的DPP-Ⅳ抑制活性[IC_(50)=(0.158±0.009)g/L]强于FAPSW[IC_(50)=(2.540±0.126)g/L];分子对接结果表明,静电相互作用、氢键、疏水作用和范德华力在FAPSW和MPGPP抑制DPP-Ⅳ活性中起重要作用;分子动力学结果表明,FAPSW和MPGPP结合DPP-Ⅳ能形成稳定的复合物结构,且MPGPP-DPP-Ⅳ复合物的稳定性要强于FAPSW-DPP-Ⅳ。结合自由能结果表明,静电相互作用在FAPSW和MPGPP结合DPP-Ⅳ中起主导作用。研究结果可为FAPSW和MPGPP在功能食品领域的开发和利用提供一定的理论参考。

虾壳源多肽酶解条件的优化及其抗冻活性和作用机制的探究

目的优化从虾壳中提取多肽的酶解条件,并探究虾壳多肽的抗冻活性及作用机制。方法以小龙虾虾壳为原料,使用复合酶酶解提取多肽。在单因素实验的基础上,通过正交实验得到虾壳最佳酶解条件。以面包酵母菌为抗冻测试对象测试其抗冻能力。采用液相色谱-串联质谱法(liquid chromatography-mass spectrometry,LC-MS/MS)对虾壳多肽的组成进行鉴定,基于多肽的理化性质和序列,结合抗冻预测平台筛选抗冻肽(antifreeze peptides,AFPs)。使用分子动力学模拟探究了AFPs的作用机制。结果在酶解温度40℃,底物质量浓度20mg/mL,加酶量12000U,酶比0.33的最佳酶解条件下,多肽提取率达到了70%以上。虾壳多肽中含有1004种肽段,能明显提升酵母菌存活率。从具有抗冻活性且置信度靠前的20条多肽中进一步筛选出了潜在的抗冻肽AFP1、AFP2。AFP2和冰面之间形成的氢键数量为14,部分水分子同时与2个氨基酸残基形成氢键,充当水桥稳定多肽的构象,AFP2与冰之间的结合能为-105.08 kcal/mol,冰结合模拟效果理想。结论AFPs与冰结合并抑制冰晶生长可能归因于带电荷的亲水性氨基酸残基与水分子形成的氢键、范德华力等分子间作用力,可开发为有益的冷冻保护剂,用于冷冻食品加工,具备良好的应用前景。

深低温保存下高效抗冻多肽的合理设计和机理探讨

The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge.Here,we propose a novel design method based on in-depth investigation of repetitive motifs in various ice-binding proteins(IBPs)with evolution analysis.In this way,several peptides with notable antifreeze activity were developed.In particular,a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition(IRI),solubility,and biocompatibility,making it suitable for use as a cryoprotective agent(CPA).A mutation analysis and molecular dynamics(MD)simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity,while the Ser18 residue can synergistically enhance their interaction with ice,revealing the antifreeze mechanism of AVD.Furthermore,to evaluate the cryoprotection potential of AVD,the peptide was successfully employed for the cryopreservation of various cells,which demonstrated significant post-freezing cell recovery.This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

鹿角多肽调控SLC7A11/GPX4轴抑制地塞米松诱导的成骨细胞铁死亡

背景:激素性股骨头坏死与成骨细胞铁死亡密切相关,鹿角多肽可以通过抑制成骨细胞铁死亡,促进成骨细胞的存活与功能的建立,具有治疗激素性股骨头坏死的潜力,但其对成骨细胞铁死亡的调控机制尚未明确。目的:探究鹿角多肽抑制地塞米松诱导成骨细胞铁死亡的作用机制。方法:①采用不同浓度梯度的鹿角多肽与地塞米松干预MC3T3-E114细胞,CCK-8法检测细胞活性,确定鹿角多肽与地塞米松的作用浓度;②采用不同质量浓度梯度鹿角多肽干预被地塞米松(800μmol/L)处理后的MC3T3-E114细胞,分为空白对照组、地塞米松组、地塞米松+鹿角多肽组,采用CCK-8法计算不同质量浓度鹿角多肽对地塞米松(800μmol/L)干预MC3T3-E114细胞增殖的影响;③借助试剂盒检测空白对照组、地塞米松组、地塞米松+鹿角多肽组中超氧化物歧化酶活性及谷胱甘肽、丙二醛、脂质过氧化物、细胞铁、活性氧含量的变化,并应用Western blot检测谷胱甘肽过氧化物酶4、溶质载体家族7成员11蛋白的表达,验证鹿角多肽抑制铁死亡的通路。结果与结论:①CCK-8法检测细胞活性,结果确定后续实验选择以鹿角多肽(10 mg/mL)和地塞米松(800μmol/L)干预MC3T3-E114细胞处理24 h;②地塞米松干预后,丙二醛、脂质过氧化物、细胞铁、活性氧含量均升高(P<0.01),谷胱甘肽含量、超氧化物歧化酶活性均降低(P<0.01),谷胱甘肽过氧化物酶4、溶质载体家族7成员11蛋白表达降低(P<0.05-0.01);鹿角多肽干预后,上述各指标变化明显被逆转(P<0.05-0.01);③结果表明,鹿角多肽可能通过调控溶质载体家族7成员11/谷胱甘肽过氧化物酶4轴抑制成骨细胞铁死亡,发挥对激素性股骨头坏死的治疗作用。

泥鳅抗氧化活性肽的制备、鉴定及其生物活性

泥鳅是亚洲常见的淡水鱼,具有较高的营养和药用价值。本研究采用短小芽孢杆菌4SP5分泌的蛋白酶水解泥鳅肉糜,超滤获得不同分子质量的水解产物。其中,分子质量小于5 ku的组分的抗氧化活性最强。经G25色谱过滤纯化,LC-MS/MS测序和鉴定过滤组分,获得6种潜在的生物活性肽。其中寡肽AFRVPTP具有良好的DPPH自由基清除活性(IC5037μg/mL)、羟自由基清除活性、超氧阴离子清除活性(IC500.73 mg/mL)。此外,该寡肽还具有抑制α-葡萄糖苷酶的作用,IC50为15.86 mg/mL。经在线预测,该寡肽无毒性和致敏性。分子对接结果表明,AFRVPTP通过与α-葡萄糖苷酶催化域的关键氨基酸结合而发挥作用,其可与Keap1活性口袋中的关键氨基酸残基(Asn387、Arg415、Tyr334、Arg380、Gln530、Tyr525、Arg483和Ser508)相互作用,表明该寡肽具有调节体内抗氧化作用的潜力。研究结果为泥鳅的精深加工提供参考依据。

大河乌猪火腿中新型α-葡萄糖苷酶抑制肽的分离、鉴定及活性研究

大河乌猪火腿在发酵和后熟过程中蛋白质降解可产生丰富的生物活性肽。为探究大河乌猪火腿中是否存在α-葡萄糖苷酶抑制肽及其活性,以大河乌猪火腿为研究对象,通过超滤分离方法制备了不同分子质量的火腿肽;以α-葡萄糖苷酶抑制率为指标,采用肽组学结合生物信息学分析方法对火腿肽进行鉴定、筛选和活性研究。结果表明:分子质量小于3 kDa的大河乌猪火腿肽具有良好的α-葡萄糖苷酶抑制活性。从大河乌猪火腿中共鉴定出143条主要来源于肌球蛋白、肌钙蛋白和β-烯醇化酶的肽序列,进一步筛选出的肽段IEEALGDK具有良好的α-葡萄糖苷酶抑制活性(IC_(50)值为1.42 mg/mL)。BIOPE P-UWM数据库检索结果显示,肽段IEEALGDK为新型的生物活性肽。肽的稳定性研究表明,肽段IEEALGDK具有较好的热稳定性、耐酸碱性和胃肠道消化稳定性。分子对接结果显示,肽段IEEALGDK主要通过氢键和疏水相互作用占据α-葡萄糖苷酶的活性残基位点Arg594、Arg727、Arg799和Arg467发挥活性作用。大河乌猪火腿的肽段IEEALGDK为新型生物活性肽,具有良好的α-葡萄糖苷酶抑制活性,研究旨在为大河乌猪火腿肽的进一步开发和利用提供理论参考。

PTEN inhibition and axon regeneration and neural repair

The intrinsic growth ability of all the neurons declines during development although some may grow better than others. Numerous intracellular signaling proteins and transcription factors have been shown to regulate the intrinsic growth capacity in mature neurons. Among them, PI3 kinase/Akt pathway is important for controlling axon elongation. As a negative regulator of this pathway, the tumor suppressor phosphatase and tensin homolog （PTEN） appears critical to con- trol the regenerative ability of young and adult neurons. This review will focus on recent research progress in axon regeneration and neural repair by PTEN inhibition and therapeutic potential of blocking this phosphatase for neurological disorders. Inhibition of PTEN by deletion in con- ditional knockout mice, knockdown by short-hairpin RNA, or blockade by pharmacological approaches, including administration of selective PTEN antagonist peptides, stimulates various degrees of axon regrowth in juvenile or adult rodents with central nervous system injuries. Im- portantly, post-injury PTEN suppression could enhance axonal growth and functional recovery in adult central nervous system after injury.

Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective:To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum.Methods:The effects of oral treatment with hydro-ethanolic extract(125,250 and 500 mg/kg)of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level,serum enzyme markers for liver function,lipid profile,total protein,serum urea,serum creatinine,and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin.In vitro assays including glucose uptake in C2 C12 cells and 3 T3-L1 adipose tissues,α-glucosidase andα-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract.DPPH and nitric oxide radical antioxidant activity of the extract was also measured.Results:The increased levels of blood glucose,triglycerides,lowdensity lipoprotein,total cholesterol,serum aspartate,and alanine transaminases,creatinine,and urea in the diabetic animals were reduced significantly(P<0.01)after treatment with Chrysophyllum albidum extract.The decreased total protein and high-density lipoprotein concentrations were normalized after treatment.In addition,the extract significantly(P<0.01)increased the transport of glucose in 3 T3-L1 cells and C2 C12 myotubes and exhibited considerable potential to inhibitα-amylase andα-glucosidase.It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals.Conclusions:Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2 C12 myotubes and 3 T3-L1 adipocytes as well as inhibiting the activities ofα-amylase andα-glucosidase.

Identifi cation and characterization of a novel tetrapeptide from enzymatic hydrolysates of Baijiu byproduct

In order to prepare angiotensin I-converting enzyme(ACE)inhibitory peptides,distilled spent grains of Chinese strong-flavor Baijiu were hydrolyzed by alcalase followed by papain under optimized conditions.A superior ACE inhibitory peptide was separated and purifi ed by ultrafi ltration and high-performance liquid chromatography(HPLC),and its amino acid sequence was further identified as Gln-Gly-Val-Pro(QGVP)by electrospray mass spectrometry(ESI-MS).QGVP formed 6 hydrogen bonds with the active site of ACE,which is responsible for reducingα-helix structure content of ACE causing subsequent inactivation.M oreover,it showed no significant cytotoxicity toward human umbilical vein endothelial cells(HUVECs),a nd signifi cantly i nduced phosphorylation of endothelial nitric oxide synthase(p-e NOS)and decreased endothelin 1(END1)expression in angiotensin I(Ang I)-treated HUVECs,demonstrating the potential antihypertensive effect.The peptide QGVP hydrolyzed from distilled spent grain proteins of Chinese strong-fl avor Baijiu was expected to be used as a food ingredient to prevent or co-treat hypertension with other chemical drugs.

Effects of heat shock protein-antigen peptide complexes (HACs) in melanoma B16 cell line on transplanted tumor development in mice

目的 :建立黑色素瘤 B16细胞热休克蛋白 -抗原肽复合物 (HACs)的制备方法并测其抑瘤效应。方法 :应用 Sephacryl S- 2 0 0凝胶过滤制备 HAC粗提物 ,应用 SDS- PAGE纯化 HACs,并测其抑瘤效应。结果 :应用 SDS- PAGE纯化的 HAC6 0、 HAC75和 HAC97不同程度地降低肿瘤发生率 ,延迟肿瘤发生时间和减慢肿瘤生长速度。结论 :6 0 0 0 0～ 970 0 0

响应面法优化香榧降血糖肽制备工艺

以香榧饼粕为原料,α-葡萄糖苷酶抑制率和蛋白水解度为主要考察指标,通过单因素实验和响应面法优化香榧降血糖肽的制备工艺。结果表明,香榧降血糖肽的最优酶解工艺参数为:碱性蛋白酶添加量10440.0 U/g,酶解pH值10.0,酶解温度46.0℃,酶解时间5.2 h,此时酶解制得蛋白水解度为24.38%的香榧降血糖肽,α-葡萄糖苷酶抑制率为59.11%。

沙棘籽粕蛋白肽的稳定性及分离纯化

以沙棘籽粕蛋白为原料,酶解制备具有抑制胰脂肪酶活性的多肽,通过超滤、大孔树脂分离、超高效液相色谱-串联质谱和分子对接对沙棘籽粕蛋白肽(seabuckthorn seed peptides from alcalase hydrolysates,SSPA)进行分离纯化、结构鉴定和筛选。以猪胰脂肪酶(porcine pancreatic lipase,PPL)抑制率为指标,稳定性分析表明,SSPA具有热稳定性和pH值稳定性,适量Na^(+)和Mg^(2+)可显著提高SSPA的PPL抑制活性,SSPA在短期贮藏过程中不会因暴露于空气而影响PPL抑制活性。超高效液相色谱-串联质谱鉴定得到31种多肽,结合分子对接筛选得到6个肽段,小于3 kDa组分中上述寡肽含量分别为VR(2.90%)、FR(7.40%)、RDR(1.10%)、APYR(1.50%)、NLLHR(1.40%)和EEAASLR(1.10%)。固相合成测定其IC_(50)值分别为VR(371.07μg/mL)、FR(243.07μg/mL)、RDR(250.50μg/mL)、APYR(350.41μg/mL)、NLLHR(220.70μg/mL)、EEAASLR(510.55μg/mL)。分子对接表明以上多肽通过氢键、π-π堆积与PPL结合。Pearson相关性分析表明,分子对接结合能与SSPA的PPL抑制率之间存在显著正相关(R^(2)=0.865,P<0.05)。

富硒核桃粕蛋白降血压肽的酶解制备及硒含量分析

以富硒核桃为原料经提油后得到富硒核桃粕,以血管紧张素转化酶(ACE)抑制作为评估标志,经单因素影响试验和响应面优化富硒核桃粕蛋白ACE抑制肽的制备工艺,并将酶解物进行超滤分离、氨基酸组成分析和硒含量测定。结果显示,富硒核桃粕蛋白ACE抑制肽的最佳酶解温度为35℃,酶解时间为2.52 h,pH值7.5、底物浓度5.39%、加酶量0.33%,在此试验条件下进行的验证试验ACE抑制率为79.13%,与理论值为78.84%较相符。最优酶解物经3 ku超滤膜分离,发现<3 ku超滤组分在1 mg/mL浓度下ACE抑制活性高达77.78%。同时,富硒核桃粕蛋白ACE抑制肽酶解物中疏水性氨基酸占27.19%,硒元素在酶解物和<3 ku超滤组分中的含量分别为0.82 mg/kg和1.35 mg/kg。该研究为食物源性补硒产品和辅助降血压健康食品的研究与开发提供了理论依据。

金华火腿中生物活性肽的抗高血压活性研究

干腌火腿在发酵成熟期间蛋白质降解可产生丰富的肽类物质。为探究干腌火腿中多肽的抗高血压活性,从金华火腿中提取多肽,通过体外检测肾素的抑制活性和动物体内灌胃实验,探究火腿多肽潜在的血压调节能力。结果显示:金华火腿多肽具有显著的肾素抑制能力,当火腿多肽质量浓度为10 mg/mL时,其肾素抑制率为62.21%。在SHR大鼠体内,当火腿多肽质量浓度为20 mg/mL时,灌胃处理组大鼠的收缩压和舒张压比未灌胃处理组显著降低(P<0.05),其血压控制效果与5 mg/mL卡托普利相近,且持续时间更长。结论:金华火腿多肽具有良好的抗高血压活性,为进一步开展干腌火腿中生物活性肽的生理调节功能研究奠定了理论基础。