摘要
蜂王幼虫是生产蜂王浆的副产物,我国蜂王幼虫资源丰富,是一种优质的昆虫蛋白质资源,长期食疗实践验证了其保健作用,开发潜力巨大。然而目前蜂王幼虫保健作用的物质基础不明确导致幼虫开发停滞在较低水平。论文以蜂王幼虫为研究对象,研究其水溶性蛋白质组成、结构和生物活性之间的构效关系,为幼虫开发提供科学依据。主要结果如下：
(1)蜂王幼虫水溶性蛋白质纯化与鉴定。对蜂王幼虫水溶性蛋白质中高丰度蛋白质和低分子量蛋白质进行鉴定,并采用离子交换、凝胶层析和反相色谱法纯化了高丰度蛋白质。结果表明,蜂王幼虫蛋白质以水溶性蛋白质为主(33.6g/100g干重),通过纯化获得两个主要蛋白质queen larvae protein-1(QLP-1)和queen larvae protein-2(QLP-2),质谱鉴定为MRJP1与其降解产物(35kDa)和MRJP2及其降解产物(40kDa)。蜂王幼虫水溶性低分子量蛋白质的分子量分布范围为5-20kDa,从中鉴定出5种蜂王浆主蛋白降解产物、3种抗氧化相关蛋白质、2种抗菌蛋白和17种幼虫发育相关蛋白质等四类蛋白质。
(2)蛋白质结构表征。QLP-1和QLP-2经过凝胶色谱分子量分布测定,分子量分别为270kDa和40kDa,native-PAGE显示均为单一条带,QLP-1和QLP-2二级结构以β-折叠为主(33%以上),a-螺旋比例较低,处于相对稳定的构象。与从蜂王浆中纯化的MRJP1和MRJP2相比,QLP-1和QLP-2的β-折叠结构比例升高,分子结构更舒展。蜂王幼虫脂类组成的结果表明,蜂王幼虫中含有丰富磷脂和辅酶Q10,蜂王幼虫和蜂王浆中甾醇的种类和含量极为相似,通过对纯化的QLP-1蛋白质中甾醇的测定,推断QLP-1和MRJP1是甾醇载体蛋白。
(3)蛋白质生物活性。采用凝胶渗透色谱和Tricine-SDS-PAGE电泳考察了QLP-2在模拟胃肠消化的水解过程,在水解度增加的同时,小分子量肽段迅速形成,水解产物ACE抑制率不断提高,酶解3h后半数抑制浓度IC50值达0.21mg/mL。采用凝胶色谱、RP-HPLC分离出一种ACE抑制肽Leu-Leu-Lys-Pro-Tyr(632.40Da),IC50值为54.9μM。同时分离出两种抗氧化肽Glu-Trp (333.14Da)和Asn-Tyr-Pro-Phe (539.24Da),对DPPH自由基清除的半数有效浓度EC50值分别为0.19和1.47mg/mL。两种活性肽不仅对ABTS和羟自由基均有很好的清除作用,还具有很强的还原力。
通过小鼠模型筛选了幼虫中具有增强免疫活性的蛋白质。灌胃给药后QLP-1显著提高正常小鼠脾指数和胸腺指数(P<0.05)；同时,QLP-1(240mg·kg-1剂量)可显著增强免疫抑制小鼠的碳廓清能力,并且使DTH小鼠耳肿胀度显著增加(P<0.05),使DTH小鼠血清中IFN-γ和TNF-α浓度显著增加(P<0.05)。结果表明QLP-1能够提高正常小鼠细胞免疫的功能,增加巨噬细胞和NK细胞的活性,诱导IFN-γ和TNF-a的产生。
Honeybee queen larvae (QL), the byproduct of royal jelly (RJ) production, are one of excellent and rich protein reservoir as a food resource in China. Long term therapeutic practice evidence indicates that QL have many beneficial effects and showed development potential in the future. However, the information about the basis of bioactive compounds has not clearly been defined yet, which lead to low utilization. The present work was therefore undertaken to explore the characterization of the water-soluble proteins in QL and investigate the relationship between the composition and the bioactivities to provide scientific basis for the utilization and development of QL.
(1) Purification and identification of water-soluble proteins from QL. High abundant proteins and low molecular weight (LMW) proteins in QL were characterized and identified by mass spectrometry. Major proteins were isolated by ion exchange, gel filtration and reverse phase chromatography. Two proteins, queen larvae protein-1(QLP-1) and queen larvae protein-2(QLP-2), were obtained, which was composed by major royal jelly protein1(MRJP1) and MRJP2as well as their degradation proteins. gel filtration chromatography (GPC) results showed that the molecular distribution of LMW proteins was between5and20kDa. The LMW proteins were dominated by their degradation proteins of MRJPs, antioxidant proteins, antibacterial proteins and proteins associated with the development and metabolism of larvae.
(2) Structure characterization of water-soluble proteins. The QLP-1and QLP-2were examined by native PAGE to verify the homogeneity of the protein. Both proteins migrated as a single diffuse band on the gel. The second structure of these proteins was dominated by β-fold (>33%), with lower content of a-helix in the relatively stable conformation. The content of β-fold structure increased compared to the MRJP1and MRJP2isolated from RJ. Results showed that little difference was observed in the sterol profiles of QL and RJ. Results showed that sterols were found to be integrated with MRJP1oligomer and QLP-1through the determination of sterols in QLP-1and QLP-2.
(3) The bioactivities of QL proteins. The major bee larvae protein was identified as QLP-2. The profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. The proteins were more digestible into peptides with molecular weights lower than3kDa. Hydrolysis capacity in intestinal digestion was considerably greater than that in gastric digestion. The angiotensin I converting enzyme (ACE) inhibitory activity of the hydrolysate increased during the hydrolysis and the IC50reached0.21mg/mL after3h digestion. An ACE inhibitory peptide was purified sequentially by gel filtration and RP-HPLC and was identified to be Leu-Leu-Lys-Pro-Tyr (632.40Da) with highest ACE inhibitory activity (IC50=54.9μM). Two antioxidant peptides were also purified and their amino acid sequences were identified as Glu-Trp (333.14Da) and Asn-Tyr-Pro-Phe (539.24Da). The median effect concentration (EC50) values of Glu-Trp and Asn-Tyr-Pro-Phe for1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical scavenging were0.19and1.47mg/mL, respectively. Two peptides also showed strong ABTS and hydroxyl radical scavenging abilities as well as reducing power.
Proteins with immune enhancing activity were screened based on the mice model. After intragastric administration of QLP-1, the spleen index and thymus index significantly increased (P<0.05), while the remaining samples had no significant effect on the normal mice thymus index (P>0.05). QLP-1(0.24g· kg-1dose) significantly enhanced the immune suppressed mice carbon clearance ability, and the mice ear swelling degree increased significantly (P<0.05). The concentration of TNF-a and IFN-y in the serum of DTH mice was significantly increased (P<0.05). The results showed that QLP-1in the larvae was the main responsible component enhancing immune function, which can improve cell immune of mouse, increase the activity of macrophages and NK cells and induce the production of TNF-a and IFN-y.
引文
Adebiyi A P, Aluko R E. Functional properties of protein fractions obtained from commercial yellow field pea (Pisum sativum L.) seed protein isolate. Food Chemistry,2011,128:902-908
Albert S, Klaudiny J, Simuth J. Molecular characterization of MRJP3, highly polymorphic protein of honeybee (Apis melliferd) royal jelly. Insect Biochemistry Molecular Biolology,1999,29(5): 427-434
Antinelli J, Zeggane S, Davico R, et al. Evaluation of (E)-10-hydroxydec-2-enoic acid as a freshness parameter for royal jelly. Food Chemistry,2003,80:85-89
Azab K S, Bashandy M, Salem M, et al. Royal jelly modulates oxidative stress and tissue injury in gamma irradiated male Wister Albino rats. North American Journal of Medical Sciences,2011, 3(6):268-276
Baggerman G, Clynen E, Huybrechts J, et al. Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry. Peptides,2003,24:1475-1485
Baldwin M A. Protein identification by mass spectrometry. Molecular and Cellular Proteomics,2004, 3:1-9
Bania J, Stachowiak D, Polanowski A. Primary structure and properties of the cathepsin G/chymotrypsin inhibitor from the larval hemolymph of Apis mellifera. European Journal of Biochemistry,1999,262:680-687
Benjakul S, Morrissey M T. Protein hydrolysates from Pacific whiting solid wastes. Journal of Agricultural and Food Chemistry,1997,45:3423-3430
Bilikova K, Hanews J, Nordhoff E, et al. Apisimin, a new serine-valine-rich peptide from honeybee (Apis mellifera L.) royal jelly:purification and molecular characterization. FEBS letters,2002, 528(1-3):125-129
Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry,1976,72:248-254
Cayusoglu K, Yapar K, Yalcin E. Royal jelly (honey bee) is a potential antioxidant against cadmium-induced genotoxicity and oxidative stress in albino mice. Journal of Medicinal Food, 2009,12(6):1286-1292
Cytrynska M, Mak P, Zdybicka-Barabas A, et al. Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. Peptides,2007,28(3):533-546
DeFoliart G R. Insects as human food:Gene defoliart discusses some nutritional and economic aspects. Crop Protection,1992,11:395-399.
DeGody I, Donahoe M, Calhoun W J, et al. Elevated TNF-a production by peripheral blood monocytes of weight losing COPD patients. American Journal of Respiratory and Critical Care Medicine,1996,153:633-637
Deigin V I, Semenets T N, Zamulaeva I A, et al. The effects of the EW dipeptide optical and chemical isomers on the CFU-S population in intact and irradiated mice. International Immunopharmacology, 2007,7:375-382
Duan X, Ocen D, Wu F, et al. Purification and characterization of a natural antioxidant peptide from fertilized eggs. Food Research International,2014,56:18-24
Elias R J, Kellerby S S, Eric, A. Antioxidant activity of proteins and peptides. Critical Reviews in Food Science and Nutrition,2008,48:430-441
Finoulst I, Pinkse M, Van Dongen W, et al. Sample preparation techniques for the untargeted LC-MS-based discovery of peptides in complex biological matrices. Journal of Biomedicine and Biotechnology,2011, article ID:245291
Fujiwara S, Imai J, Fujiwara M, et al. A potent antibacterial protein in royal jelly:purification and determination of the primary structure of royalisin. The Journal of Biological Chemistry,1990,265, 11333-11337
Furusawa T, Rakwal R, Nam H W, et al. Comprehensive royal jelly (RJ) proteomics using one-and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins. Journal of Proteome Research,2008,7,3194-3229
Gomez-Ruiz J A, Ramos M, Recio I. Angiotensin-converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion. International Dairy Journal,2004,14:1075-1080
Grimble G K. The significance of peptides in clinical nutrition. Annual Review of Nutrition,1994,14: 419-447.
Guo H, Kouzuma Y, Yonekura M. Structures and properties of antioxidative peptides derived from royal jelly protein. Food Chemistry,2009,113:238-245
Gu Y, Majumder K, Wu, J. QSAR-aided in silico approach in evaluation of food proteins as precursors of ACE inhibitory peptides. Food Research International,2011,44:2465-2474
Guang C, Phillips R D. Plant food-derived Angiotensin I converting enzyme inhibitory peptides. Journal of Agricultural and Food Chemistry,2009,57:5113-5120
Hattori N, Nomoto H, Fukumitsu H, et al. Royal jelly and its unique fatty caid, 10-hydroxy-trans-2-decenoic acid, promote neurogenesis by neural stem/progenitor cells in vitro. Biomedical Research,2007,28(5):261-266
Huang C Y, Chi L L, Huang W J, et al. Growth stimulating effect on queen bee larvae of histone deacetylase inhibitors. Journal of Agricultural and Food Chemistry,2012,60:6139-6149
Hur S J, Lim B O, Decker E A, et al. In vitro human digestion models for food applications. Food Chemistry,2011,125:1-12
Jones P J, MacDougall D E, Ntanios F Y, et al. Dietary phytosterols as cholesterol-lowering agents in humans. Canadian Journal of Physiology and Pharmacology,1997,75:217-227
Kamakura M. Royalactin induces queen differentiation in honeybees. Nature,2011,473:478-483
Kamakura M, Fukuda T, Fukushima M, et al. Storage-dependent degradation of 57-kDa protein in royal jelly:a possible marker for freshness. Bioscience, Biotechnology and Biochemistry,2001a, 65:277-284
Kamakura M, Mitani N, Fukuda T, et al. Antifatigue effect of fresh royal jelly in mice. Journal of Nutritional Science and Vitaminology,2001b,47(6):394-401
Kamakura M, Suenobu N, Fukushima M. Fifty-seven-kDa protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum. Biochemical and Biophysical Research Communications,2001c,282:865-874
Karadeniz A, Simsek N, Karakus E, et al. Royal jelly modulates oxidative stress and apoptosis in liver and kidneys of rats treated with cisplatin. Oxidative Medicine and Cellular Longevity,2011, article ID:981793
Kim S, Wijesekara I. Development and biological activities of marine-derived bioactive peptides:a review. Journal of Functional Foods,2010,2:1-9
Kimura Y, Takaku T, Okuda H. Antitumor and antimetastatic actions by royal jelly in Lewis lung carcinoma-bearing mice. Journal of Traditional Medicines,2003,20(5):195-200
Kodai T, Umebayashi K, Nakatani T, et al. Compositions of Royal Jelly II. Organic Acid Glycosides and Sterols of the Royal Jelly of Honeybees (Apis mellifera). Chemical and Pharmaceutical Bulletin,2007,55(10):1528-1531
Kramer K J, Childs C N, Speirs R D, et al. Purification of insulin-like peptides from insect haemolymph and royal jelly. Insect Biochemistry,1982,12:91-98
Lai Y P, Gallo R L. AMPed up immunity:how antimicrobial peptides have multiple roles in immune defense. Trends in Immunology,2009,30:131-141
Larre C, Lupi R, Gombaud Q et al. Assessment of allergenicity of diploid and hexaploid wheat genotypes:Identification of allergens in the albumin/globulin fraction.2011,74(8):1279-1289
Lee S, Choi J, Park S, et al. Determination of phospholipids in soybean Glycine max (L.Merr) cultivars by liquid chromatography-tandem mass spectrometry. Journal of Food Composition and Analysis,2010,23:314-318
Lee Y J, Kang S J, Kim B M, et al. Cytotoxicity of honeybee (Apis mellifera) venom in normal human lymphocytes and HL-60 cells. Chemico-Biological Interactions,2007,169,189-197
Li J, Feng M, Zhang L, et al. Proteomics analysis of major royal jelly protein changes under different storage conditions. Journal of Proteome Research,2008,7:3339-3353
Li J, Wang T, Zhang Z, et al. Proteomic analysis of royal jelly from three strains of western honeybees (Apis mellifera). Journal of Agricultural and Food Chemistry,2007,55:8411-8422
Li J, Wu J, Rundassa D B, et al. Differential protein expression in honeybee (Apis mellifera L.) larvae: underlying caste differentiation. PLoS one,2010,5(10):1-11
Li T S C, Beveridge T H J, Drover J C G Phytosterol content of sea buckthorn (Hippophae rhamnoides L.) seed oil:extraction and identification. Food Chemistry,2007,101:1633-1639
Liu X, Zhang M, Jia A, et al. Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum. Food Research International,2013,50: 339-343
Lo W M, Li-Chan E C. Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein. Journal of Agricultural and Food Chemistry,2005,53: 3369-3376
Majtan J, Kovacova E, Bilikova K, et al. The immunostimulatory effect of the recombinant apalbumin 1-major honeybee royal jelly protein-on TNFa release. International Immunopharmacology,2006, 6:269-278
Majtan J, Kumar P, Majtan T, et al. Effect of honey and its major royal jelly protein 1 on cytokine and MMP-9 mRNA transcripts in human keratinocytes. Experimental Dermatology,2010,19:73-79
Marambe H K, Shand P J, Wanasundara J P. Release of angiotensin Ⅰ-converting enzyme inhibitory peptides from flaxseed (Linum usitatissimum L.) protein under simulated gastrointestinal digestion. Journal of Agricultural and Food Chemistry,2011,59:9596-9604
McLay R N, Pan W, Kastin A J. Effects of peptides on animal and human behavior:a review of studies published in the first twenty years of the journal Peptides. Peptides,2001,22:2181-2255
Meisel H. Biochemical properties of regulatory peptides derived from milk proteins. Biopolymers, 1997,43:119-128
Miguel M, Alonso M J, Salaices M, et al. Antihypertensive, ACE-inhibitory and vasodilator properties of an egg white hydrolysate:effect of a simulated intestinal digestion. Food Chemistry,2007,104: 163-168
Mizutani Y, Shibuya Y, Takahashi T, et al. Major royal jelly protein 3 as a possible allergen in royal jelly-induced anaphylaxis. The Journal of Dermatology,2011,38(11):1079-1081
Moreaua R A, Whitakerb B D, Hicksa K B. Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Progress in Lipid Research, 2002,41:457-500
Moure A, Dominguez H, Parajo J C. Antioxidant properties of ultrafiltration-recovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochemistry,2006,41:447-456
Nagai T, Inoue R. Preparation and the functional properties of water extract and alkaline extract of royal jelly. Food Chemistry,2004,84:181-186
Nalinanon S, Benjakul S, Kishimura H. Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna(Thunnus alalunga). Food Chemistry,2010,121:49-55
Nozaki R, Tamura S, Ito A, et al. A rapid method to isolate soluble royal jelly proteins. Food Chemistry,2012,134:2332-2337
Noda N, Umebayashi K, Nakatani T, et al. Isolation and characterization of some hydroxy fatty and phosphoric acid esters of 10-hydroxy-2-decenoic acid from the royal jelly of honeybees (Apis mellifera). Lipids,2005,40(8):833-838
Okamoto I, Taniguchi Y, Kunikata T, et al. Major royal jelly protein 3 modulates immune responses in vitro and in vivo. Life Sciences,2003,73:2029-2045
Panchaud A, Affolter M, Kussmann M. Mass spectrometry for nutritional peptidomics:How to analyze food bioactives and their health effects. Journal of proteomics,2012,75:3546-3559
Power O, Jakeman P, FitzGerald R J. Antioxidative peptides:enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids,2013,44:797-820
Puchalska P, Marina M L, Garcia M C. Isolation and identification of antioxidant peptides from commercial soybean-based infant formulas. Food Chemistry,2014,148:147-154
Ramadan M F, Al-Ghamdi A. Bioactive compounds and health-promoting properties of royal jelly:A review. Journal of Functional Foods,2012,4:39-52
Rao S, Sun J, Liu Y, et al. ACE inhibitory peptides and antioxidant peptides derived fromin vitro digestion hydrolysate of hen egg white lysozyme. Food Chemistry,2012a,135:1245-1252
Rao S, Ju T, Sun J, et al. Purification and characterization of angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysate of hen egg white lysozyme. Food Research International,2012b,46:127-134
Ratcliffe N A, Mello C B, Garcia E S, et al. Insect natural products and processes:new treatments for human disease. Insect Biochemistry and Molecular Biology,2011,41:747-769
Rho S J, Lee J, Chung Y I, et al. Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract. Process Biochemistry,2009,44:490-493
Romanelli A, Moggio L, Montella R C, et al. Peptides from Royal Jelly:Studies on the antimicrobial activity of jelleins, jelleins analogs and synergy with temporins. Journal of Peptide Science,2011, 17(5):348-352
Sampath Kumar N S, Nazeer R A, Jaiganesh R. Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein. Peptides,2011,32: 1496-1501
Sante-Lhoutellier V, Engel E, Aubry L, et al. Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility. Meat Science,2008,79:777-783
Santos K S, dos Santos L D, Mendes M A, et al. Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.), Insect Biochemistry Molecular Biology,2005,35:85-91
Sarmadi B H, Ismail A. Antioxidative peptides from food proteins:A review. Peptides,2010,31: 1949-1956
Schagger H. Tricine-SDS-PAGE. Nature Protocols,2006,1(1):16-22
Schmitz O, Brock B, Rungby J. Amylin agonists:a novel approach in the treatment of diabetes. Diabetes,2004,53(S3):S233-S238
Schmitzova J, Klaudiny J, Albert S. A family of major royal jelly proteins of the honeybee Apis mellifera L. Cell Molecular Life Science,1998,54:1020-1030
Semina O V, Semenets T N, Zamulaeva I A, et al. Effects of iEW synthetic peptide isomers on bone marrow colony-forming capacity in vivo. Bulletin of Experimental Biology and Medicine,2005, 140:348-351
Shen L, Zhang W, Jin F, L et al. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line. Journal of Agricultural and Food Chemistry,2010,58:9190-9197
Shen F, Albert S, Su S, et al. Comparative analysis of C-terminal repetitive sequences of MRJP2 among six species of the genus Apis. Acta Entomologica Sinica,2007,50(10):1049-1056
Simuth J, Bilikova K, Kovacova E, et al. Immunochemical approach to detection of adulteration in honey:Physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey. Journal of Agricultural and Food Chemistry,2004,52:2154-2158
Soulier P, Farines M, Soulier J. Triterpene alcohols,4-methylsterols and 4-desmethylsterols of sal and illipe butters. Journal of the American Oil Chemists' Society,1990,67:388-393
Suzuki K M, Isohama Y, Maruyama H, et al. Estrogenic activities of fatty acids and a sterol isolated from royal jelly. Evidence-Based Complementary and Alternative Medicine,2008,5:295-302
Sultana A, Nurunnabi A H M, Nasir U M, et al. A dipeptide YY derived from royal jelly proteins inhibits renin activity. International Journal of Molecular Medicine,2008,21:677-681
Tamura S, Amano S, Kono T, et al. Molecular characteristics and physiological functions of major royal jelly protein 1 oligomer. Proteomics,2009a,9:5534-5543
Tamura S, Kono T, Harada C, et al. Estimation and characterisation of major royal jelly proteins obtained from the honeybee Apis merifera. Food Chemistry,2009b,114:1491-1497,
Tang C, Wang X. Physicochemical and structural characterisation of globulin and albumin from common buckwheat(Fagopyrum esculentum Moench) seeds. Food Chemistry,2010,121:119-126
Taniguchi Y, Kohnoa K, Inoue S I, et al. Oral administration of royal jelly inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice. International Immunopharmacology,2003,3: 1313-1324
Takaki-Doi S, Hashinoto K, Yamamura M, et al. Antihypertensive activities of royal jelly protein hydrolysate and its fractions in spontaneously hypertensive rats. Acta Medica Okayama,2009, 63(1):57-64
Tavares T, Contreras M M, Amorim M, et al. Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme:in vitro effect and stability to gastrointestinal enzymes. Peptides,2011,32:1013-1019
Terada Y, Narukawa M, Watanabe T. Specific hydroxy fatty acids in royal jelly activate TRPA1. Journal of Agricultural and Food Chemistry,2011,59:2627-2635
Tetsuya K, Kazue U, Takafumi N, et al. Compositions of royal jelly Ⅱ. Organic acid glycosides and sterols of the royal jelly of honeybees(Apis mellifera). Chemical and Pharmaceutical Bulletin, 2007,55:1528-1531
Terashima M, Oe M, Ogura K, et al. Inhibition strength of short peptides derived from an ACE inhibitory peptide. Journal of Agricultural and Food Chemistry,2011,59(20):11234-11237
Tokunaga K H, Yoshida C, Suzuki K M, et al. Antihypertensive effect of peptides from royal jelly in spontaneously hypertensive rats. Biological and Pharmaceutical Bulletin,2004,27(2):189-192
Townsend G F, Morgan J F, Tolnai S, et al. Studies on the in vitro:antitumor activity of fatty acids Ⅰ. 10-hydroxy-2-decenoic Acid from royal jelly. Cancer Research,1960,20:503
Udenigwe C C, Lu Y, Han C, et al. Flaxseed protein-derived peptide fractions:antioxidant properties and inhibition of lipopolysaccharide-induced nitric oxide production in murine macrophages. Food Chemistry,2009,116:277-284
Vanderplanck M, Michez D, Vancraenenbroeck S, et al. Micro-quantitative method for analysis of sterol levels in honeybees and their pollen loads. Analytical Letters,2011,44:1807-1820
Vercruysse L, Van Camp J, Smagghe G ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein:a review. Journal of Agricultural and Food Chemistry,2005,53(21): 8106-8115
Vucevic D, Melliou E, Vasilijic S, et al. Fatty acids isolated from royal jelly modulate dendritic cell-mediated immune response in vitro. International Immunopharmacology,2007,7:1211-1220
Wu Z, Pan D, Zhen X, et al. Angiotensin I-converting enzyme inhibitory peptides derived from bovine casein and identified by MALDI-TOF-MS/MS. Journal of the Science of Food and Agriculture,2013,93:1331-1337
Xu X, Dong J, Mu X, et al. Supercritical CO2 extraction of oil, carotenoids, squalene and sterols from lotus (Nelumbo nucifera Gaertn) bee pollen. Food and Bioproducts Processing,2011,89:47-52
Xu X, Gao Y. Radical scavenging activity of sea buckthorn oils from different parts of sea buckthorn Berry. Food Science and Biotechnology,2009,18:312-316
Yang B, Karlsson R M, Oksman P H, et al. Phytosterols in sea buckthorn (Hippophae rhamnoides L.) berries:Identification and effects of different origins and harvesting Times. Journal of Agricultural and Food Chemistry,2001,49:5620-5629
Yang Y, Marczak E D, Yokoo M, et al. Isolation and antihypertensive effect of angiotensin I-converting enzyme (ACE) inhibitory peptides from spinach rubisco. Journal of Agricultural and Food Chemistry,2003,51:4897-4900
Yoshida H, Tomiyama Y, Tanaka M, et al. Distribution of fatty acids in triacylglycerols and phospholipids from peas (Pisum sativum L.). Journal of the Science of Food and Agriculture,2007, 87:2709-2714
You S J, Wu J. Angiotensia-Ⅰ converting enzyme inhibitory and antioxidant activities of egg protein hydrolysates produced with gastrointestinal and nongastrointestinal enzymes. Journal of Food Science,2011,76:C801-807
Zhan Y, Wang L, Liu J, et al. Choline plasmalogens isolated from swine liver inhibit hepatoma cell proliferation associated with caveolin-1/Akt signaling. PLoS one,2013,8(10):e77387
Zhang H Y. Structure-activity relationships and rational design strategies for radical-scavenging antioxidants. Current Computation-Aided Drug Design,2005,1:257-273
包郁明,侯虎,乔若瑾,等.皱纹盘鲍脏器糖蛋白结构的初步鉴定及其免疫活性研究.中国海洋药物杂志,2012,31(4)：20-24
岑宁,孙鸿才,黄双源.蜂王胎抗衰作用研究初报.中国养蜂,1999,50(1)：10
蔡柳,林亲录.蜂王浆的研究进展.中国食物与营养,2007,8：19-22
陈建,李建科.王浆高产蜜蜂与喀尼鄂拉蜂幼虫期蛋白质组比较.中国农业科学,2008,41(10)：3292-3299
陈惠娟,廖森泰,刘吉平.昆虫蛋白资源的利用研究概况.广东农业科学,2011,19：105-108
陈茂彬,黄琴,干信,等.植物甾醇油酸酯对荷瘤小鼠免疫调节作用研究.武汉理工大学学报,2005,3：46-48
程佳宙,贾月亮,林庆敏,等.蜂王浆对小鼠免疫功能影响.贵阳医学院学报,2007,32(6)：632-635
段博文,李运,刘昕,等.柳茶多糖对小鼠免疫功能的影响.中国中药杂志,2010,35(11)：1466-1469
丁美会.中华蜜蜂王浆抗菌肽Acc-royalisin原核表达及抑菌活性研究：[硕士学位论文].杭州：浙江大学,2010
董贺,张太平,彭士明,等.山楂谷甾醇的提取及降血脂和增强免疫的研究.天然产物研究与开发,2009,5：60-63
何伟,徐响,孙丽萍,等.油菜、菊花和荷花蜂花粉中磷脂的色谱分析.食品科学,2011,32(18)：185-189
胡熠凡,陈忠周,田文礼,等.蜂王浆中主蛋白成分(MRJP2)的分离纯化及圆二色谱分析.食品科学,2013,34(23)：57-61
金庆武,周瑞华.蜂王幼虫干粉蛋白质的生物利用率和营养食效性研究.中国自然医学杂志,2003,12(5)：249-251
李永利,张焱.高效液相色谱法测定保健食品中脑磷脂.中国卫生检验杂志,2007,17(5)：843
李娟,赵萍,代方银,等.家蚕血液胰凝乳蛋白酶抑制剂的多态性分布.昆虫学报,2006,4：550-555
李爽,王静,张颖.蜂王幼虫蛋白质提取工艺研究.食品工业科技,2006,6：128-129
刘芳,苏松坤.中华蜜蜂王浆蛋白MRJP5的结构分析与功能预测.蜜蜂杂志,2011,5：1-4
李奕冉,姜玉新,李朝品.昆虫蛋白对动物免疫功能影响研究进展.动物医学进展,2010,31(9)：81-84
刘合生.蜂王浆活性蛋白的分离、纯化、生物活性及结构初步表征：[硕士学位论文].武汉：华中农业大学,2009
刘秀娟,吴珍红,缪晓青.蜂王幼虫酶解工艺的研究.食品研究与开发,2006,27(7)：136-137
刘玮玮,冯磊.植物甾醇降血脂作用研究进展.浙江医学,2008,30(2)：202-204
马仁公.蜂王幼虫、工蜂幼虫和雄蜂蛹的新用途.中国蜂业,2007,58(5)：32-33
倪彬,陈丰,刘建伟,等.蜂王幼虫粉延缓衰老和抗应激能力的实验研究.营养学报,2002,24(4)：398-400
况炜,陈慧玲,章皓,等.羊栖菜多糖免疫调节活性的实验研究.实用医学杂志,2012,28(23)：3872-3873
潘建国,郑尧隆,段怡,等.蜂王胎中-10-羟基-癸烯酸含量的测定.养蜂科技,2004,4：2-4
庞广昌,陈庆森,胡志和,等.蛋白质的消化吸收及其功能评述.食品科学,2013,34(9)：375-391
彭友瑞,钟方旭,杨博,等.蜂王浆对D-半乳糖衰老模型大鼠学习记忆行为的影响.食品科学,2011.32(15)：269
单媛媛,马美湖,黄茜,等.红外光谱法研究温度变化对卵粘蛋白构象的影响.高等学校化学学报,2012,33(9)：1950-1956
沈星灿,梁宏,何锡文,等.圆二色光谱分析蛋白质构象的方法及研究进展.分析化学,2004,3：388-394
沈平锐.蜂皇胎对幼龄大白鼠生长影响的实验简报.蜜蜂杂志,1990,05：8
沈平锐,罗光华.蜂王幼虫的营养食效性及其在食品中的开发利用.食品科学,1993,3：63-65
王静凤,傅佳,王玉明,等.革皮氏海参皂苷对小鼠免疫功能的调节作用.中国海洋大学学报,2010,40(2)：28-32
汪宁,朱荃,周义维,等.蜂王浆冻干粉对HepG2细胞葡萄糖消耗作用及对糖尿病小鼠降糖作用的实验研究.食品科学,2006,27(9)：233-235
王先梅,赵连友,郑强荪,等.糜酶抑制剂对自发性高血压大鼠心肌纤维化的影响.心脏杂志,2006,1：31-34
吴黎明,周群,周骁,等.蜂王浆不同贮存条件下蛋白质二级结构的Fourier变换红外光谱研究.光谱学与光谱分析,2009,129(11)：82-87
吴炜亮,吴国杰,梁道双,等.ACE抑制肽的生理功能和研究进展.现代食品科技,2006,22(3)：251-254
许建香,张智武,刘永东,等.RP-HPLC法测定蜂王浆水溶性蛋白及其相关产物对ACE的抑制作用.食品与发酵工业,2008,9：124-127
许喜兰.蜂王幼虫的营养与医疗保健作用.养蜂科技,2003,5：37
徐响,孙丽萍,董捷.蜂王浆中脂类脂肪酸组成的研究.食品科学,2009,30(14)：213-214
杨宇清,魏伟.儿茶素对环磷酰胺诱导小鼠迟发性超敏反应的影响.中国新药杂志,2007,23：1940-1943
易建勇,董鹏,王永涛,等.应用SRCD和FTIR分析超高压处理对蘑菇多酚氧化酶二级结构的影响.光谱学与光谱分析,2012,32(2)：317-323
曾星凯,谢国秀,吴小波,等.蜂王浆活性组分癸烯酸和氨基酸含量变化对小白鼠抗疲劳作用研究.江西农业大学学报,2009,31(3)：526-529
颜伟玉,曾星凯,谢国秀,等.蜂王浆中不同活性组分对大鼠降血脂效果影响.江西农业大学学报,2009,31(5)：826-829
殷客卿,杨寒冰,陈晶,等.蜂王幼虫的营养成分分析.蜜蜂杂志,2010,7：3-6
岳兵.蜂王幼虫酶解物成分分析及活性评价：[硕士学位论文].北京：中国农业科学院,2010
岳兵,薛晓锋,吴黎明,等.高效液相色谱法测定蜂王幼虫中辅酶Q10含量.食品科学,2010,31(10)：206-208
曾志将,吴小波,张飞,等.蜂王浆机械化生产关键技术研究与应用(Ⅰ)-仿生免移虫生产器设计.江西农业大学学报,2013,4：842-847
翟爱华,黄明海,贾建,等.蜂幼虫分离蛋白提取研究.粮油食品科技,2004,12(2)：9-10
张鸿,吴玉荷.类维生素物质-辅酶Q10的研究进展.国外医学卫生学分册,2002,29(6)：370- 373
张智武,单斌,彭文君,等.蜂王浆中水溶性蛋白质和活性肽的研究进展.食品研究与开发,2007,28(1)：32-36
周瑞华,裴银辉,刘学义,等.蜂王幼虫对小鼠免疫功能的影响及机理探讨.食品科学,1999,11：11-13
周金慧,薛晓峰,张金振,等.高效液相色谱法测定蜂王幼虫中的几丁多糖含量.2008,59(10)：21-22
周瑞华,裴银辉,刘学义,等.蜂王幼虫对小鼠免疫功能的影响及机理探讨.食品科学,1999,1：11-13