WO2016090563A1 - 芋螺毒素衍生物、其制备方法和抗氧化应用 - Google Patents

芋螺毒素衍生物、其制备方法和抗氧化应用 Download PDF

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WO2016090563A1
WO2016090563A1 PCT/CN2014/093417 CN2014093417W WO2016090563A1 WO 2016090563 A1 WO2016090563 A1 WO 2016090563A1 CN 2014093417 W CN2014093417 W CN 2014093417W WO 2016090563 A1 WO2016090563 A1 WO 2016090563A1
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snail
antioxidant
seq
antioxidant peptide
peptide
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PCT/CN2014/093417
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French (fr)
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莫芬
林志龙
闻博
童婷
刘杰
杜朝钦
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深圳华大基因研究院
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Priority to PCT/CN2014/093417 priority patent/WO2016090563A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links

Definitions

  • the present application relates to the field of antioxidant peptides, and in particular to an antioxidant peptide derived from the snail, and a preparation method and application of the antioxidant peptide.
  • free radicals In the metabolism of biological organisms, a class of atoms or molecules with high oxidative activity and one or several unpaired electrons is usually produced, which are called free radicals. It mainly includes superoxide anion (O 2 -.), hydroxyl radical (.OH), singlet oxygen (lO 2 ) and hydrogen peroxide (H 2 O 2 ) [1] . Free radicals have unpaired electrons that are very prone to loss or electrons and have super-active chemistry.
  • Antioxidant peptide is a kind of natural antioxidant. It is generally composed of 2-20 amino acid residues. It can inhibit the peroxidation of biomacromolecules and remove excess free radicals in the body. It has high biological activity and safety in human food.
  • the industrial, cosmetic and pharmaceutical industries have great potential for development.
  • There are three main ways for antioxidant peptides to achieve antioxidant activity [3-4] one is to chelate metal ions; the other is to act as a hydrogen donor or an electron donor to stabilize free radicals; the third is to promote the decomposition of peroxides, such as promotion.
  • antioxidant peptides There are three main sources of antioxidant peptides: 1. Naturally occurring bacteria, fungi and antioxidant peptides in animals and plants, ie endogenous antioxidant peptides, but these peptides are less in content, high in extraction, purification and purification; Degradation of food-derived proteins, these active peptides have high safety and mild production conditions, but it is difficult to achieve efficient enrichment of specific active peptides and release of specified sequences; 3.
  • Directional chemical synthesis of antioxidant peptides, application of peptide synthesis technology New peptides can be designed to carry out research on structure-activity relationships, with the purpose of synthesizing specific sequences Peptides to achieve efficient enrichment of active ingredients.
  • the synthesis method is limited by the equipment investment and high product cost.
  • the technical application is usually limited to the synthesis of small molecule oligopeptides. Thanks to the optimization of chemical solid phase synthesis peptide technology, the synthesis of small molecule peptides is currently easier to implement.
  • the snail is mainly grown in tropical waters. It usually lives in the warm sea. It belongs to the Mollusca, Gastropoda, Prosobranchia, Neogastropoda. , Conidae, Conus.
  • Conopeptide (Conotoxin, CTX) is a kind of biologically active peptide toxoid obtained from the snail. It has a relatively low molecular weight, is rich in disulfide bonds, is structurally stable, has high activity, is highly selective, and is easy to synthesize. The advantages have been extensively studied. It is estimated that there are about 700-1000 Cone snails in the world [6] , and each snail has at least 50-200 active peptides in its venom.
  • conotoxin is a "rich mining area" to be excavated. China has discovered about 100 species of snails, mainly distributed in the Nansha Islands, the Xisha Islands, Hainan Island and the waters near Taiwan, and a few are distributed along the coasts of Guangdong and Guangxi.
  • the purpose of the present application is to provide a novel snail antioxidant peptide, and a preparation method and application of the snail antioxidant peptide.
  • the present application discloses a snail antioxidant peptide, and the snail antioxidant peptide of the present application is derived from a snail, and the snail antioxidant peptide is a polypeptide of the formula
  • Equation 1 XC n -X'-CC-X"
  • X consists of 1-2 amino acids, the 1-2 amino acids contain at least one hydrophilic amino acid, 0-1 hydrophobic amino acids; X' consists of 5-12 amino acids; X" consists of 0-3 Amino acid composition, in the 0-3 amino acids, when X" is 1-3 amino acids, containing 1-2 hydrophobic amino acids and 0-1 hydrophilic amino acids; C represents cysteine, n is equal to 1 Or 2 indicates that there is one or two cysteines.
  • the snail antioxidant peptides of the present application are all isolated and identified from the snails of the snails, and, after research and analysis, the polypeptides separated from the snails of the snails are less than or equal to the number of amino acids. And having at least one hydrophilic amino acid at the X-terminus as shown in Formula 1, and having at least one hydrophobic amino acid in the X′ terminal, and having cysteine at both ends, this type
  • the polypeptide has antioxidative activity; therefore, the snail antioxidant peptide of the formula shown in the formula 1 is proposed.
  • the formula shown in the formula 1 is limited to the polypeptide isolated from the snail, and does not guarantee the conformity of the other formula. Polypeptides also possess antioxidant properties; however, the formula shown in Formula 1 can be used as a reference for screening antioxidant peptides from polypeptides from other sources.
  • the amino acid sequences of seven spiro antioxidant peptides are sequentially Seq ID No.1 to Seq ID No.7. Display sequence
  • Seq ID No. 1 RCCVHPACHDCICCIT
  • Seq ID No. 2 RCCSVSICQPPPVCECCA;
  • Seq ID No. 3 RCCSQDCRVCIPCCPY
  • Seq ID No. 4 RPCCPRDTWCCGFP
  • Seq ID No. 5 KCCTMSVCQPPPVCTCCA;
  • Seq ID No. 6 RCRCEQTCGTCVPCC
  • Seq ID No. 7 SLCCPEDRWCC.
  • the snail antioxidant peptide of the present application is derived from the barrel snail and can be directly isolated and extracted from the snail; however, since the snail antioxidant peptide of the present application has a simple structure and is easy to synthesize, It can be obtained directly by artificial synthesis. It should be noted that the seven snail antioxidant peptides of the present application are all isolated and identified from the barrel snail by the same method, and the antioxidant effects of the seven snail antioxidant peptides are similar, and both are far superior to the present.
  • Some antioxidant peptides in the embodiment of the present application, the existing antioxidant peptide glutathione is specifically used as a comparison, and the results show that the IC50 value of the seven snail antioxidant peptides of the present application can be higher than that of the glutathione.
  • the glycopeptide is reduced by 84%, and the lowest is also reduced by 75%, and the antioxidant activity is greatly superior to glutathione.
  • the present application discloses the use of the snail antioxidant peptide of the present application for scavenging free radicals, including DPPH radicals, hydroxyl radicals, singlet oxygen, and At least one of hydrogen peroxide and superoxide anion radicals.
  • the snail antioxidant peptide of the present application has the basic function of an antioxidant peptide, and, in the implementation of the present application, the snail antioxidant peptide of the present application has an activity compared with the existing glutathione.
  • the invention has the advantages of stronger radical scavenging effect and simple structure and easy synthesis. Therefore, the snail antioxidant peptide of the present application can be more conveniently and widely applied in the fields of food, health care products, cosmetics and medicines.
  • the conopeptides of the present application are all derived from the genus Barracuda, and the amino acid sequence of the snail antioxidant peptide is the sequence shown in any one of Seq ID No. 1 to Seq ID No. 7.
  • the seven sequences are all isolated and identified in the barrel snail, and the polypeptides of any of the sequences shown in the present invention have antioxidant properties, can effectively remove free radicals, and take DPPH free radicals as an example to remove the effects. It is better than existing glutathione and can be applied to free radical scavenging and food, health care products, cosmetics and pharmaceuticals related to free radical scavenging.
  • the other side of the application also discloses a preparation method of the snail antioxidant peptide of the present application, which comprises performing reductive alkylation of dithiothreitol and iodoacetamide on the conotoxin extracted from the snail
  • the sample is subjected to peptide mass spectrometry detection, and the mass spectrometry data is analyzed by data analysis and biological information analysis to obtain the amino acid sequence of the snail antioxidant peptide, and finally the chemical synthesis of the snail antioxidant peptide is carried out.
  • these chemically synthesized conopeptides are specifically seven polypeptides which independently exhibit antioxidant activity in the sequence of Seq ID No. 1 to Seq ID No. 7.
  • the mass spectrometric detection of the polypeptide is carried out by using a nanoliter high performance liquid chromatography-mass spectrometer.
  • conch anti-oxidation peptide composition comprising at least one of seven conopeptides, an amino acid sequence of seven conopeptides The sequence is in the order of Seq ID No. 1 to Seq ID No. 7.
  • the seven snail antioxidant peptides of the sequence shown by Seq ID No. 1 to Seq ID No. 7 were obtained;
  • Each of the snail antioxidant peptides has good free radical scavenging properties and can be used alone.
  • two or more conopeptides can also be used in combination; therefore, the conopeptide composition of the present application is resistant to the seven species of snails found in the present application. At least one composition of the peptides.
  • the present application discloses the use of a snail antioxidant peptide composition for scavenging free radicals, including DPPH radicals, hydroxyl radicals, singlet oxygen, and At least one of hydrogen peroxide and superoxide anion radicals.
  • each conopeptide is prepared by the preparation method disclosed in the present application.
  • the snail antioxidant peptide of the present application has high activity, low dose and high safety compared with the conventional antioxidant peptide.
  • the antioxidant peptide activity was calculated by DPPH free radical scavenging rate of 50%.
  • the IC50 value of the snail antioxidant peptide of the present application is up to 0.043 mM, and the lowest is 0.027 mM; and the IC50 value of glutathione is 0.169 mM;
  • the applied snail antioxidant peptide can be up to 84% lower than glutathione, and the antioxidant effect is much better than glutathione.
  • the snail antioxidant peptide of the present application has a simple structure, is easy to be artificially synthesized, and can effectively scavenge free radicals, and lays a foundation for the widespread application of antioxidant peptides in food, health care products, cosmetics and pharmaceuticals.
  • Figure 1 is a graph showing the change trend of DPPH clearance rate of the polypeptide CP-btl01 in the examples of the present application;
  • Figure 2 is a graph showing the trend of DPPH clearance change of the polypeptide CP-btl02 in the examples of the present application;
  • Figure 3 is a graph showing the trend of DPPH clearance rate change of the polypeptide CP-btl03 in the examples of the present application;
  • Figure 4 is a graph showing the change trend of DPPH clearance rate of the polypeptide CP-btl04 in the examples of the present application;
  • Figure 5 is a graph showing the trend of DPPH clearance change of the polypeptide CPTx-btl01 in the examples of the present application.
  • Figure 6 is a graph showing the trend of DPPH clearance change of the polypeptide CPTx-btl02 in the examples of the present application.
  • Figure 7 is a graph showing the trend of DPPH clearance change of the polypeptide CPTx-btl04 in the examples of the present application.
  • Fig. 8 is a graph showing the trend of changes in DPPH clearance rate of the positive control GSH in the examples of the present application.
  • Conotoxin is a class of biologically active peptide toxoids, and has been reported in many studies; however, the study of conotoxin or antioxidant peptides derived from conus snails is relatively lacking. Nazeer et al. showed that anti-oxidation peptides can be obtained by enzymatic hydrolysis of the snails of the snails [7] . This application is based on this study, assuming that the conotoxin secreted by the barrel snails may also contain Antioxidant peptides, combined with high-throughput mass spectrometry, found and identified the natural antioxidant peptides present in the barrel conotoxin. Further, seven conopeptides of the sequence shown by Seq ID No. 1 to Seq ID No. 7 were proposed.
  • the seven kinds of snail antioxidant peptides of the present application are all extracted from the same barrel-shaped snail body by the same method, and can be prepared in large quantities by artificial synthesis method because of its simple structure;
  • the snail antioxidant peptides have good antioxidant effects and can be used singly or in combination, and are not specifically limited in the present application.
  • antioxidant activity depends on the main factors such as the molecular weight of the polypeptide, amino acid composition and amino acid sequence [8] .
  • the nucleophilic sulfur-containing amino acids Cys, Met, acidic amino acids Asp, Glu, aromatic amino acids Trp, Tyr, Phe and the like are more active.
  • the N-terminus or C-terminus of the polypeptide contains a hydrophobic amino acid, it can contribute to the interaction between the antioxidant peptide and the fatty acid, and can enhance the antioxidant activity.
  • IC50 or IC50 value in the present application refers to the amount of antioxidant peptide used in the case of a DPPH clearance of 50% in millimoles per liter (mmol/L, abbreviated mM).
  • DPPH or DPPH free radical means 1,1-diphenyl-2-trinitrophenylhydrazine.
  • the conopeptides treated as described above were enriched on a Strata-X C18 column.
  • the sample is applied to the Strata-X column in a low concentration acetonitrile environment, and the peptide is bound to the column by reverse phase reaction; then the column is washed with a low concentration of acetonitrile solution to remove impurities in the peptide sample that cannot be combined with the strata-X column, such as Inorganic salt, etc.; after salt removal, the peptide bound to the column is eluted with high concentration of acetonitrile, so that the salt and peptide in the peptide sample can be obtained. Separation.
  • Strata-X C18 enrichment operation was performed according to standard procedures: 1) 1 ml methanol was added to activate the column; 2) 1 ml 0.1% FA equilibrium column was added; 3) venom sample was loaded 1 ml, buffer (5% ACN + 0.1% FA) was washed, The wash was repeated 3 times; 4) 100% ACN elution, and the eluate was collected.
  • the enriched polypeptide was examined for molecular weight by MALDI-TOF-MS.
  • the 240 ug mixed peptide was subjected to fractionation by SCX-HPLC (Shimadzu) system, buffer A: 10 mM KH 2 PO 4 in 25% ACN, pH 3.5, and buffer B contained 500 mM potassium chloride on the basis of buffer A.
  • the flow rate was 1 ml/min, 0-40% linear binary gradient buffer B was eluted for 10 minutes, 40-90% buffer B was eluted for 2 minutes, 90% buffer B was eluted for 3 minutes, and absorbance was detected at 214 nm.
  • a total of 10 fractions were collected by gradient elution.
  • the collected fractions were desalted by a C18 solid phase extraction column (Strata-X, Phenomenex) and reconstituted with 30 ⁇ l of 0.1% formic acid for nanoLC-MS/MS analysis.
  • the LC/MS instrument is based on Shimadzu's nano HPLC chromatograph system and AB Sciex's Triple TOF 5600 mass spectrometer system.
  • Each of the pre-isolated polypeptide fractions was separately separated by a conventional 12 cm long, 75 ⁇ m inner diameter, and an integral capillary analysis column packed with a Welch Materials brand XB-C18 column having a pore size of 3 ⁇ m and a pore size of 300 nl/min.
  • the injection volume was detected to be 25 ⁇ l, and the elution gradient was that the concentration of liquid B was uniformly increased from 5% to 45% for 40 minutes.
  • the mass spectrometry collected electrospray voltage was 2.5 kV
  • the auxiliary gas pressure was 30 PSI
  • the sheath gas pressure was 15 PSI
  • the source temperature was 150 °C.
  • the acquisition of the first-order mass spectrum uses a high-resolution mode greater than or equal to 30,000.
  • the valence state of the parent ion is selected from the range of 2 to 5 charges.
  • 30 secondary mass spectrometry fragments can be continuously performed, thus completing 30 secondary spectral iones in 250 ms. The scan produces more than 120 secondary spectra per second, with a total cycle time of 3.3 seconds.
  • the original mass spectrometry data detected by nanoLC-MS/MS was formatted into MGF and then searched and identified by Mascot search software.
  • polypeptide sequence seven polypeptides as shown in Table 1 were obtained by sequence characteristic analysis for chemical synthesis.
  • the specific feature analysis includes 86 of the existing reports and patents in the statistics of this application.
  • sequence characteristics of the antioxidant peptides as well as experimental verification. It should be noted that in this experiment, a large number of polypeptide fragments were obtained in the experimental stage, and seven polypeptides having antioxidant activity shown in Table 1 were screened by experiments, and the specific experimental verification was as follows. Recorded.
  • the linear peptide of snail was synthesized by solid phase chemical synthesis of methoxycarbonyl (Fmoc), which was completed by Shanghai Jill Biochemical Co., Ltd.
  • the ability to scavenge free radicals is generally determined by in vitro chemical methods.
  • the reaction mechanism of DPPH free radical scavenging activity is the scavenging ability of free radicals, which is usually expressed by the scavenging rate.
  • DPPH free radical scavenging activity can be expressed by IC50, ie The concentration of the antioxidant when the DPPH free radical scavenging rate reaches 50%.
  • IC50 concentration of the antioxidant when the DPPH free radical scavenging rate reaches 50%.
  • chemical antioxidant capacity of antioxidant peptides was evaluated by the IC50 value of DPPH. The specific method is as follows.
  • the DPPH used in this example was DPPH dissolved in 95% ethanol at a concentration of 0.2 mmol/L. 2.0 mL of DPPH solution was placed in a test tube, 2.0 mL of polypeptide was added, and the mixture was shaken and shaken. After reacting for 60 min in a dark room at room temperature, if precipitation occurred, centrifugation was performed at 6000 rpm for 15 min, and the supernatant was taken at 517 nm to measure its absorbance (Ai).
  • the DPPH clearance rate of the seven antioxidant peptides of the present application is much better than that of the GSH positive control.
  • the CP-btl03 sample has the highest IC50 dosage of 84% lower than that of GSH, and the radical scavenging effect is excellent.
  • the seven antioxidant peptides of the present application can achieve a clearance rate of about 80% at 0.06-0.08 mM, and about 0.35 mM for GSH to achieve the same clearance rate.
  • the seven antioxidant peptides of the present application can effectively scavenge DPPH free radicals, and the scavenging effect is better than that of the positive control reduced glutathione GSH; and the antioxidant peptide of the present application has a simple structure, is easy to be artificially synthesized, can be mass-produced, and is widely used. In the fields of food, health products, cosmetics and pharmaceuticals.
  • the peptide drug of this example is derived from the barrel snail, which has a clear structure and a clear mechanism of action. Compared with general organic small molecule drugs, it has high activity, small dosage, low toxicity, and metabolic end products are amino acids. Advantages; compared with proteins, the smaller polypeptides are almost non-immunogenic; they can be chemically synthesized, with high purity and controllable quality; therefore, they have higher safety.

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Abstract

提供的是芋螺毒素衍生物、其制备方法和抗氧化应用。还提供的是包括该芋螺毒素衍生物的食品、保健品、化妆品和药品。

Description

一种芋螺抗氧化肽及其制备方法和应用 技术领域
本申请涉及抗氧化肽领域，特别是涉及一种源自于芋螺的抗氧化肽，以及该抗氧化肽的制备方法和应用。
背景技术
在生物有机体的新陈代谢过程中通常会产生一类具有高度氧化活性、带有一个或几个不配对电子的原子或分子，这些都被叫做自由基。主要包括超氧阴离子(O2-.)、羟自由基(.OH)、单线态氧(lO2)和过氧化氢(H2O2)[1]。自由基带有未成对的电子，非常易于失去或者得到电子，具有超活泼的化学性质。当机体内自由基产生过多，清除失衡时，过多的自由基将使生物膜上不饱和脂肪酸发生超氧化，导致细胞内外离子转运紊乱、红细胞溶血、线粒体功能紊乱等，引起细胞结构和功能的紊乱，科学研究同时也发现氧化与很多常见的疾病有着密不可分的关系[2]。目前为止，医学界发现很多退化性疾病如动脉粥样硬化、肿瘤、白内障、辐射损伤、烧伤、衰老、肺病、肝病等都与自由基有关。
近年来，国内外对生物活性肽的关注日趋升温，已成为食品、保健品、药品等产业的研究热点。抗氧化肽是天然抗氧化剂的一种，一般由2-20个氨基酸残基组成，能抑制生物大分子过氧化和清除机体内过量自由基，具有较高的生物活性和安全性，在人类食品工业，化妆品及医药行业具有巨大发展潜力。抗氧化肽实现抗氧化活性主要有三种方式[3-4]：一是螯合金属离子；二是作为供氢体或供电子体来稳定自由基；三是促进过氧化物的分解，如促进双氧水的分解。发现较早的天然存在于动植物体内的抗氧化肽有谷胱甘肽(γ-Glu-Cys-Gly)、肌肽(β-alanyl-L-histidine)、鹅肌肽(β-alanyl-L-1-methylhistidine)和蛇肉肽(β-alanyl-L-3-Methylhistidine)等[5]。其中的还原型谷胱甘肽就是通过供电子使自由基失活，保护机体细胞免受自由基损伤，分子内巯基是其发挥生物学功能的主要官能团。
抗氧化肽的来源主要有以下三种方式：1.天然存在细菌，真菌及动植物体内的抗氧化肽，即内源性抗氧化肽，但这些肽含量较少，提取分离纯化成本高；2.食源性蛋白的降解，这些活性肽安全性高，生产条件温和，但难以实现特定活性肽段的高效富集及指定序列的释放；3.定向化学合成的抗氧化肽，应用多肽合成技术可以设计新的多肽来开展构效关系的研究，目的性的合成特定序列的多 肽，实现有效成分的高效富集。不过合成法受设备投资大、产品成本高等条件限制，其技术应用通常限于小分子寡肽的合成，得益于化学固相合成多肽技术的优化，小分子多肽的合成目前较易实现。
芋螺主要生长于热带海域，一般多生活在暖海，在生物分类学上属于软体动物门(Mollusca)，腹足纲(Gastropoda)，前鰓亚纲(Prosobranchia)，新腹足目(Neogastropoda)，芋螺科(Conidae)，芋螺属(Conus)。芋螺毒素(Conopeptide，Conotoxin，CTX)是从芋螺中获得的一类具有生物活性的多肽类毒素，具有相对分子量小、富含二硫键、结构稳定、高活性、高选择性及易于合成的优势已被广泛研究。据估计，全球约有700-1000种芋螺(Cone snails)[6]，每种芋螺的毒液中至少含有50-200种活性肽，最近的研究结果显示活性肽种类可能更多[6]。也就是说，理论上有20万种甚至更多的活性肽存在于芋螺毒素中，因此，芋螺毒素是一待挖掘的“富矿区”。我国现今发现了约100佘种芋螺，主要分布在南沙群岛、西沙群岛、海南岛及台湾附近海域，少数分布在广东、广西沿海。
参考文献：
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[2]Wiseman，H.and Halliwell，B.Damage to DNA by reactive oxygen and nitrogen species：role in in□ammatory disease and progression to cancer[J].Biochem.J.1996，313：17 29.
[3]Suetsuna，K.，Ukeda，H.，&Ochi，H.Isolation and characterization of free radical scavenging activities peptides derived from casein[J].J.Nutr.Biochem.2000，11：128□131.
[4]Saiga，A.，Tanabe，S.&Nishimura，T.Antioxidant activity of peptides obtained from porcine myofibrillar protein by protease treatment[J].J.Agric.Food Chem.2003，51：3661□3667.
[5]Chan K M，Decker E A.Endogenous muscle antioxidants[J].Critical Reviews in Food Science and Nutrition，1994，34：403□426.
[6]朱翠，李力，戴秋云.几类新家族芋螺毒素研究进展[J].中国海洋药物，2014，33(2)：84-89。
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发明内容
本申请的目的是提供一种新的芋螺抗氧化肽，以及芋螺抗氧化肽的制备方法和应用。
为了实现上述目的，本申请采用了以下技术方案：
本申请一方面公开了一种芋螺抗氧化肽，本申请的芋螺抗氧化肽源自于桶形芋螺，芋螺抗氧化肽为式一所示通式的多肽，
式一：X-Cn-X’-CC-X”
其中，X由1-2个氨基酸组成，该1-2个氨基酸中含有至少一个亲水性氨基酸，0-1个疏水性氨基酸；X’由5-12个氨基酸组成；X”由0-3个氨基酸组成，该0-3个氨基酸中，当X”为1-3个氨基酸时，含有1-2个疏水性氨基酸和0-1亲个水性氨基酸；C表示半胱氨酸，n等于1或2，表示有一个或两个半胱氨酸。
需要说明的是，本申请的芋螺抗氧化肽都是从桶形芋螺中分离鉴定的，并且，经过研究分析显示，分离自桶形芋螺的多肽中，其满足氨基酸个数小于或等于18，且在如式一所示的X端含有至少一个亲水性氨基酸，X”端中其不为0时，含有至少一个疏水性氨基酸，并且两端具有半胱氨酸，这种类型的多肽具备抗氧化活性；因此，提出式一所示通式的芋螺抗氧化肽。该式一所示通式仅限于分离自桶形芋螺的多肽，不保证其它来源的符合式一通式的多肽也具备抗氧化性；但是式一所示通式可以作为从其它来源的多肽中筛选抗氧化肽的参考依据。
在以上研究的基础上，本申请具体公开了七个抗氧化效果较好的芋螺抗氧化肽，七个螺抗氧化肽的氨基酸序列依序为Seq ID No.1至Seq ID No.7所示序列；
Seq ID No.1：RCCVHPACHDCICCIT；
Seq ID No.2：RCCSVSICQPPPVCECCA；
Seq ID No.3：RCCSQDCRVCIPCCPY；
Seq ID No.4：RPCCPRDTWCCGFP；
Seq ID No.5：KCCTMSVCQPPPVCTCCA；
Seq ID No.6：RCRCEQTCGTCVPCC；
Seq ID No.7：SLCCPEDRWCC。
需要说明的是，本申请的芋螺抗氧化肽源自于桶形芋螺，可以直接从桶形芋螺中分离提取；但是，由于本申请的芋螺抗氧化肽结构简单、易于合成，也 可以采用人工合成方式直接获得。还需要说明的是，本申请的七个芋螺抗氧化肽都是采用相同的方法分离鉴定自桶形芋螺，并且七个芋螺抗氧化肽的抗氧化效果相近，且都远优于现有的抗氧化肽；本申请的实施方式中特别采用了现有的抗氧化肽谷胱甘肽作为对比，结果显示，本申请的七个芋螺抗氧化肽的IC50值浓度最高可以比谷胱甘肽减小84％，最低也可以减小75％，抗氧化活性极大的优于谷胱甘肽。
在本申请的芋螺抗氧化肽的基础上，本申请公开了本申请的芋螺抗氧化肽在清除自由基中的应用，这些自由基包括DPPH自由基、羟基自由基、单线态氧、过氧化氢和超氧阴离子自由基中的至少一种。
可以理解，由于本申请的芋螺抗氧化肽具备抗氧化肽的基本功能，并且，在本申请的实现方式中，与现有的谷胱甘肽相比，本申请的芋螺抗氧化肽具备更强的自由基清除效果，并且，结构简单、易于合成，因此，本申请的芋螺抗氧化肽可以更方便且广泛的应用于食品、保健品、化妆品和药品等领域。
本申请的芋螺抗氧化肽，都源自于桶形芋螺，芋螺抗氧化肽的氨基酸序列为Seq ID No.1至Seq ID No.7任一项所示序列。该七个序列都是分离鉴定于桶形芋螺，其任一项所示序列的多肽都具备抗氧化性，能够实现对自由基的有效清除，并且以清除DPPH自由基为例，清除效果都比现有的谷胱甘肽更好，可以应用于自由基清除，以及与自由基清除相关的食品、保健品、化妆品和药品等领域。
本申请的另一面还公开了本申请的芋螺抗氧化肽的制备方法，该制备方法包括，对从桶形芋螺中提取的芋螺毒素进行二硫苏糖醇和碘乙酰胺还原烷基化处理，然后对样品进行多肽质谱检测，对质谱检测数据进行数据解析和生物信息分析，获得芋螺抗氧化肽的氨基酸序列，最后采用化学合成芋螺抗氧化肽。本申请的优选方案中，这些化学合成的芋螺抗氧化肽具体为Seq ID No.1至Seq ID No.7所示序列的七个独立发挥抗氧化活性的多肽。
优选的，本申请的制备方法中，多肽质谱检测采用纳升高效液相色谱-质谱联用仪进行。
本申请的另一面公开了一种芋螺抗氧化肽组合物，该芋螺抗氧化肽组合物中含有七种芋螺抗氧化肽中的至少一种，七种芋螺抗氧化肽的氨基酸序列依序为Seq ID No.1至Seq ID No.7所示序列。
需要说明的是，本申请通过对桶形芋螺的毒素进行分析研究，获得了Seq ID No.1至Seq ID No.7所示序列的七个芋螺抗氧化肽；经试验证实，这七个芋螺抗氧化肽都具有良好的自由基清除性能，可以单独使用，在一些特殊的需求中， 如食品、保健品、化妆品或药品中，也可以两个或多个芋螺抗氧化肽组合使用；因此，本申请的芋螺抗氧化肽组合物是由本申请所发现的七种芋螺抗氧化肽中的至少一种组成。
在本申请的芋螺抗氧化肽组合物的基础上，本申请公开了芋螺抗氧化肽组合物在清除自由基中的应用，自由基包括DPPH自由基、羟基自由基、单线态氧、过氧化氢和超氧阴离子自由基中的至少一种。
本申请的芋螺抗氧化肽组合物中，各芋螺抗氧化肽采用本申请公开的制备方法制备。
本申请的有益效果在于：
本申请的芋螺抗氧化肽，与传统的抗氧化肽相比，具有高活性，低剂量和高安全性。以DPPH自由基清除率50％计算抗氧化肽活性，本申请的芋螺抗氧化肽IC50值最高为0.043mM，最低可达到0.027mM；而谷胱甘肽的IC50值为0.169mM；可见，本申请的芋螺抗氧化肽最高可以比谷胱甘肽低84％，抗氧化效果极大优于谷胱甘肽。本申请的芋螺抗氧化肽结构简单、易于人工合成，并且能有效清除自由基，为抗氧化肽在食品、保健品、化妆品和药品等领域的广泛应用奠定了基础。
附图说明
图1：是本申请实施例中多肽CP-btl01的DPPH清除率变化趋势分析图；
图2：是本申请实施例中多肽CP-btl02的DPPH清除率变化趋势分析图；
图3：是本申请实施例中多肽CP-btl03的DPPH清除率变化趋势分析图；
图4：是本申请实施例中多肽CP-btl04的DPPH清除率变化趋势分析图；
图5：是本申请实施例中多肽CPTx-btl01的DPPH清除率变化趋势分析图；
图6：是本申请实施例中多肽CPTx-btl02的DPPH清除率变化趋势分析图；
图7：是本申请实施例中多肽CPTx-btl04的DPPH清除率变化趋势分析图；
图8：是本申请实施例中阳性对照GSH的DPPH清除率变化趋势分析图。
具体实施方式
芋螺毒素是一类具有生物活性的多肽类毒素，已经有比较多的研究报道；但是，芋螺毒素或源自于芋螺的抗氧化肽的研究相对缺乏。Nazeer等的研究显示，桶形芋螺其内脏处通过酶解可以获得抗氧化多肽[7]；本申请正是在该研究的基础上，假设桶形芋螺分泌的芋螺毒素中也可能含有抗氧化多肽，并结合高通量质谱技术发现并鉴定出了桶形芋螺毒素中存在的天然抗氧化多肽。并进一步 的提出了Seq ID No.1至Seq ID No.7所示序列的七种芋螺抗氧化肽。
需要说明的是，本申请的七种芋螺抗氧化肽都是采用同样的方法从相同的桶形芋螺主体中提取的，由于其结构简单，也可以采用人工合成方法大量制备；并且，七种芋螺抗氧化肽都具有很好的抗氧化效果，可以单独使用，也可以组合在一起使用，在本申请中不做具体限定。
现有的抗氧化肽活性构效关系研究通常认为，抗氧化活性取决于多肽的分子量、氨基酸组成和氨基酸序列等主要因素[8]。20种氨基酸的活性存在较大差异，其中活性较高的有亲核性含硫氨基酸Cys、Met，酸性氨基酸Asp、Glu，芳香族氨基酸Trp、Tyr、Phe等。同时当多肽中的N端或者C端包含疏水性氨基酸，能够助于抗氧化肽与脂肪酸之间的相互作用，可增强抗氧化活性。本申请统计了已有报道文献和专利中的86条抗氧化肽的序列特性；发现抗氧化多肽序列长度集中在2-10和15左右，净电荷多分布在-2和2之间，等电点则主要分布在2-6和10左右。N端氨基酸出现频率高于10％的有Leu和Tyr，C端氨基酸频率高于10％的有Lys。以上统计作为本申请进行生物信息分析、筛选抗氧化肽的依据。
本申请中IC50或IC50值是指DPPH清除率为50％时的抗氧化肽用量，单位为毫摩尔每升(mmol/L，简写mM)。DPPH或DPPH自由基是指1，1-二苯基-2-三硝基苯肼。
下面通过具体实施例和附图对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。
实施例一 芋螺多肽的制备
1、芋螺多肽提取及还原烷基化
将4只海南产桶形芋螺砸壳后解剖剪取毒管，收集芋螺毒液。使用Bradford法测定毒液中蛋白浓度为6.48mg/ml。取总蛋白量为0.5mg，加入终浓度为1mM的二硫苏糖醇(简写，DTT)，于56℃反应1h，还原冷却至室温后加入终浓度为55mM的碘乙酰胺(简写，IAM)于室温暗室反应45min。
2、多肽富集
将经上述处理的芋螺多肽，用Strata-X C18柱进行富集。在低浓度乙腈环境下上样品到Strata-X柱，肽段通过反相作用与柱料结合；之后用低浓度乙腈溶液继续洗柱子除去肽段样品中无法与strata-X柱结合的杂质，如无机盐等；除盐后，用高浓度乙腈将柱料上结合的肽段洗脱下来，使肽段样品中的盐分与肽段得以 分离。Strata-X C18富集操作按标准规程操作：1)加1ml甲醇活化柱子；2)加1ml 0.1％FA平衡柱子；3)毒液样品上样1ml，buffer(5％ACN+0.1％FA)wash，重复wash 3次；4)100％ACN洗脱，收集洗脱液。富集多肽用MALDI-TOF-MS检测其分子量大小。
实施例二 芋螺抗氧化多肽的制备
1、芋螺多肽序列鉴定
240ug混合肽经SCX-HPLC(Shimadzu)系统进行组分分离，buffer A：10mM KH2PO4in 25％ACN，pH 3.5，buffer B所含成分是在buffer A的基础上含500mM氯化钾。流速1ml/min，0-40％的线性二元梯度的buffer B洗脱10分钟，40-90％的buffer B洗脱2分钟，90％的buffer B洗脱3分钟，214nm进行吸光度检测，通过梯度洗脱共收集10个馏分。收集馏分经C18固相萃取柱除盐(Strata-X，Phenomenex)后用30μl的0.1％的甲酸复溶，进行nanoLC-MS/MS分析。
2、nanoLC-MS/MS分析
液质联用仪采用的是岛津的nano HPLC色谱仪系统和AB Sciex的Triple TOF 5600质谱仪系统。每个预分离好的多肽组分分别经过自制的12cm长，75μm内径，填充了粒径3μm孔径120A的Welch Materials品牌XB-C18柱料的Ultimate毛细管分析柱分离，流速为300nl/min。检测进样体积为25μl，洗脱梯度为B液浓度从5％均匀上升到45％经过40min。质谱采集的电喷雾电压为2.5kV，辅气气压为30PSI，鞘气气压为15PSI，源温度为150℃。一级质谱的采集使用大于或等于30000的高分辨率模式。二级质谱的采集，选择母离子价态在2电荷到5电荷的范围，扫描一次一级质谱后可接着连续做30个二级质谱碎裂，这样在250ms内完成对30个二级谱子离子的扫描，每秒可产生超过120张二级谱，总的一个循环时间为3.3秒。
3、数据分析
将nanoLC-MS/MS检测得到的原始质谱数据进行格式转换成MGF后再用Mascot搜索软件进行数据搜索鉴定。具体包括根据转录组测序构建的蛋白数据库进行搜索，参数设置如下：肽段容差：-tol 10-tolu ppm；二级质谱中碎片离子的质量误差：-itol 0.02-itolu Da；固定修饰：-mods＝′Carbamidomethyl(C)′；酶-cle None；-decoy＝1；电荷数：-charge＝′2+，3+and 4+′；允许最大漏切数：-pfa 0；可变修饰：-it_mods＝′Oxidation(M)，Gln-＞pyro-Glu(N-term Q)′；instrument＝ESI-FTICR。得到的多肽序列中，通过序列特征分析获得如表1所示的七个多肽进行化学合成。具体的特征分析包括，本申请统计的已有报道文献和专利中的86 条抗氧化肽的序列特性，以及实验验证。需要说明的是，本例在进行试验阶段，获得了大量的多肽片段，表1所示的七个具备抗氧化活性的多肽，是经过实验验证筛选出来的，具体的实验验证如后面实施例所记载。
表1 抗氧化肽序列
名称 序列 分子量 Seq ID No.
CP-btl01 RCCVHPACHDCICCIT 1775.69道尔顿 1
CP-btl02 RCCSVSICQPPPVCECCA 1896.76道尔顿 2
CP-btl03 RCCSQDCRVCIPCCPY 1847.71道尔顿 3
CP-btl04 RPCCPRDTWCCGFP 1639.66道尔顿 4
CPTx-btl01 KCCTMSVCQPPPVCTCCA 1872.72道尔顿 5
CPTx-btl02 RCRCEQTCGTCVPCC 1660.61道尔顿 6
CPTx-btl04 SLCCPEDRWCC 1313.47道尔顿 7
4、多肽合成
应用芴甲氧羰基(Fmoc)固相化学合成的方法合成芋螺线性肽，由上海吉尔生化合成公司完成。
实施例三 多肽清除DPPH试验
因抗氧化测定方法机理的差异性，使得测定结果可能存在差异性，清除自由基能力的测定一般采用体外化学法，利用体系产生自由基或本身就是自由基，通过反应体系物理化学特性的变化间接表征自由基的清除能力，实验易于操作，可靠性高。DPPH自由基清除活性的反应机理是对自由基的清除能力，通常采用清除率来表示，由于DPPH自由基清除率与抗氧化剂的浓度通常成正比，因此DPPH自由基清除活性又可用IC50表示，即当DPPH自由基清除率达到50％时，抗氧化剂的浓度值。本例以DPPH的IC50值评价抗氧化肽的化学抗氧化力。具体方法如下。
本例采用的DPPH为溶于95％乙醇的浓度为0.2mmol/L的DPPH。将2.0mL DPPH溶液置于试管中，加入2.0mL多肽，振荡混匀，室温暗室反应60min后，若出现沉淀，6000rpm离心15min，取上清液在517nm处测其吸光值(Ai)，空白为2.0mL 95％乙醇加入2.0mL蒸馏水调零，对照为2.0mL DPPH溶液加上2.0mL蒸馏水在测定波长下的吸光值(Ac)，酶解液在测定波长的吸光值为Aj。 以实施例二合成的七个多肽进行试验，并选取天然抗氧化还原型谷胱甘肽(GSH)做为阳性对照，每个样品设计5个浓度，计算各个浓度下的清除率，并绘制标准曲线；分别计算每个样品在清除率达到50％时的用量，即IC50值，用以评价样品的抗氧化能力。清除率计算公式如下：
DPPH清除率(％)＝[1-(Ai-Aj)÷Ac]×100
本例测量的七个多肽和GSH阳性对照的IC50如表2所示，绘制的标准曲线如图1至图8所示。
表2 各样品的IC50值
  CP-btl01 CP-btl02 CP-btl03 CP-btl04 CPTx-btl01 CPTx-btl02 CPTx-btl04 GSH
IC50(mM) 0.030 0.037 0.027 0.043 0.032 0.028 0.035 0.169
由表2可见，本申请的七个抗氧化肽的DPPH清除率都大大优于GSH阳性对照，如CP-btl03样品，其IC50的用量最高可以比GSH低84％，自由基清除效果优异。从图1-8也可以看出，本申请的七个抗氧化肽在0.06-0.08mM时，清除率可以达到80％左右；而GSH则需要0.35mM左右，才能达到相同的清除率。本申请的七种抗氧化肽能够有效清除DPPH自由基，清除效果优于阳性对照还原型谷胱甘肽GSH；并且本申请的抗氧化肽结构简单，易于人工合成，可以大批量生产，广泛应用于食品、保健品、化妆品和药品等领域。
此外，本例的肽类药物源于桶形芋螺，其结构清楚，作用机制明确，与一般有机小分子药物相比，具有活性高、用药剂量小、毒副作用低、代谢终产物为氨基酸等优点；而与蛋白类相比，较小的多肽几乎没有免疫原性；可化学合成，产品纯度高，质量可控；因此具备更高的安全性。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明，不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本申请的保护范围。

Claims (16)

  1. 一种芋螺抗氧化肽，其特征在于：所述芋螺抗氧化肽源自于桶形芋螺，芋螺抗氧化肽为式一所示通式的多肽，
    式一：X-Cn-X’-CC-X”
    其中，X由1-2个氨基酸组成，该1-2个氨基酸中含有至少一个亲水性氨基酸，0-1个疏水性氨基酸；X’由5-12个氨基酸组成；X”由0-3个氨基酸组成，该0-3个氨基酸中，当X”为1-3个氨基酸时，含有1-2个疏水性氨基酸和0-1亲个水性氨基酸；C表示半胱氨酸，n等于1或2，表示有一个或两个半胱氨酸。
  2. 根据权利要求1所述的芋螺抗氧化肽，其特征在于：所述芋螺抗氧化肽具体为Seq ID No.1至Seq ID No.7所示序列的七个独立发挥抗氧化活性的多肽；
    Seq ID No.1：RCCVHPACHDCICCIT；
    Seq ID No.2：RCCSVSICQPPPVCECCA；
    Seq ID No.3：RCCSQDCRVCIPCCPY；
    Seq ID No.4：RPCCPRDTWCCGFP；
    Seq ID No.5：KCCTMSVCQPPPVCTCCA；
    Seq ID No.6：RCRCEQTCGTCVPCC；
    Seq ID No.7：SLCCPEDRWCC。
  3. 根据权利要求1或2所述的芋螺抗氧化肽在清除自由基中的应用，所述自由基包括DPPH自由基、羟基自由基、单线态氧、过氧化氢和超氧阴离子自由基中的至少一种。
  4. 根据权利要求1或2所述的芋螺抗氧化肽在食品、保健品、化妆品和药品中的应用。
  5. 一种芋螺抗氧化肽，其特征在于：所述芋螺抗氧化肽源自于桶形芋螺，芋螺抗氧化肽的氨基酸序列为Seq ID No.1至Seq ID No.7任一项所示序列。
  6. 根据权利要求5所述的芋螺抗氧化肽在清除自由基中的应用，所述自由基包括DPPH自由基、羟基自由基、单线态氧、过氧化氢和超氧阴离子自由基中的至少一种。
  7. 根据权利要求5所述的芋螺抗氧化肽在食品、保健品、化妆品和药品中的应用。
  8. 根据权利要求1、2或5所述的芋螺抗氧化肽的制备方法，其特征在于：所述制备方法包括，对从所述桶形芋螺中提取的芋螺毒液进行二硫苏糖醇和碘乙酰胺还原烷基化处理，然后进行多肽质谱检测，对质谱检测数据进行搜索鉴定序列和生物信息分析，获得芋螺抗氧化肽的氨基酸序列，最后采用化学合成所述芋螺抗氧化肽。
  9. 根据权利要求8所述的制备方法，其特征在于：所述多肽质谱检测采用纳升高效液相色谱-质谱联用仪进行。
  10. 一种芋螺抗氧化肽，其特征在于：所述芋螺抗氧化肽采用以下方法制备，从桶形芋螺中提取的芋螺毒液，对芋螺毒液进行二硫苏糖醇和碘乙酰胺还原烷基化处理，然后进行多肽质谱检测，对质谱检测数据进行搜索鉴定序列和生物信息分析，获得芋螺抗氧化肽的氨基酸序列，最后采用化学合成所述氨基酸序列的芋螺抗氧化肽；化学合成的芋螺抗氧化肽具体为Seq ID No.1至Seq ID No.7所示序列的七个独立发挥抗氧化活性的多肽。
  11. 根据权利要求10所述的芋螺抗氧化肽，其特征在于：所述多肽质谱检测采用纳升高效液相色谱-质谱联用仪进行。
  12. 根据权利要求10或11所述的芋螺抗氧化肽在清除自由基中的应用，所述自由基包括DPPH自由基、羟基自由基、单线态氧、过氧化氢和超氧阴离子自由基中的至少一种。
  13. 根据权利要求10或11所述所述的芋螺抗氧化肽在食品、保健品、化妆品和药品中的应用。
  14. 一种芋螺抗氧化肽组合物，其特征在于：所述芋螺抗氧化肽组合物中含有七种芋螺抗氧化肽中的至少一种，所述七种芋螺抗氧化肽的氨基酸序列依序为Seq ID No.1至Seq ID No.7所示序列。
  15. 根据权利要求14所述的芋螺抗氧化肽在清除自由基中的应用，所述自由基包括DPPH自由基、羟基自由基、单线态氧、过氧化氢和超氧阴离子自由基中的至少一种。
  16. 根据权利要求14所述的芋螺抗氧化肽在食品、保健品、化妆品和药品中的应用。
PCT/CN2014/093417 2014-12-10 2014-12-10 芋螺毒素衍生物、其制备方法和抗氧化应用 WO2016090563A1 (zh)

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