梅花鹿茸多肽的化学结构及生物活性

在马鹿茸活性多肽结构与功能研究基础上, 从新鲜梅花鹿茸中分离纯化了活性单体多肽, 确定了其化学结构, 并与马鹿茸多肽进行结构与活性比较. 利用离子交换层析、 凝胶过滤层析及反相高效液相色谱层析等生物化学技术, 从梅花鹿茸中分离得到1个新多肽, SDS-PAGE电泳显示为一条带, HPLC图谱为单一峰, MALDI-TOF MS给出该多肽的精确分子量为3263.4, 其等电点pI=8.15. 一级结构研究表明, 该多肽是由32个氨基酸残基组成的直链多肽, 不含半胱氨酸, 富含缬氨酸、 赖氨酸、 亮氨酸和甘氨酸, 氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. 生物活性检测结果表明, 该多肽可促进原代培养的表皮细胞和软骨细胞增殖, 也能刺激NIH3T3成纤维细胞株的分裂. 梅花鹿茸多肽与马鹿茸多肽在结构上均为32个氨基酸残基组成的直链多肽, 但第5, 8, 11和30位氨基酸残基不同. 2种多肽结构上的变化并未影响其促细胞增殖生物活性.     

梅花鹿茸中活性多肽的纯化、测序及功能研究

利用凝胶过滤、离子交换层析及C18反相柱层析等方法, 从梅花鹿茸中分离得到了一个多肽CNT14, SDS-PAGE电泳显示其为一条带, HPLC图谱为单峰, 激光解析电离飞行时间质谱测定其分子量为1479.9028. 氨基酸组成分析结果表明, 此多肽主要含缬氨酸、亮氨酸、谷氨酸及脯氨酸, 用电喷雾串联质谱法测序结果为E-P-T-V-L-D-E-V-C-L-A-H-G-P. 活性检测结果表明, 该多肽能够明显促进小鼠海马HT22细胞增殖. 同时, 采用固相合成法对该多肽进行了合成, 与所分离的天然多肽进行了结构与性质的比较.     

Comparison of Structure and Biological Activity of Natural Polypeptide from Velvet Antlers of Cervus elaphus with Those of Synthesized Polypeptide

Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and reversed-phase high-performance liquid chromatography(RP-HPLC) were used to isolate and purify the natural polypeptide from velvet antler(nVAP) of Cervus elaphus(C. elaphus), which has a molecular weight of 3215.8 and the primary structure of VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM. The homology of the protein sequence in nVAP with known protein sequence is less than 50%, suggesting that nVAP appears to be a new bioactive substance. At a level of 0.4―50 μg/mL, nVAP promotes mitosis in epidermal cells, chondrocytes and NIH3T3 fibroblasts primarily cultured in a significant way. Given that a yield of high-purity nVAP isolated from C. elaphus is 0.001%, nVAP is artificially synthesized to prepare synthetic velvet antler polypeptide(sVAP) according to its primary structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of sVAP shows a single band, and its HPLC spectrum displays a single peak. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) was used to identify sVAP to be of a molecular weight of 3200 and the consistency between primary structures of sVAP and nVAP. Bioactivity test shows that at a dose of 5―40 μg/mL, sVAP promotes the proliferation of primarily cultured epidermal cells and NIH3T3 cell line. From the traditional Chinese medicine theory, velvet antler from Cervus nippon(C. nippon) and velvet antler from C. elaphus are considered as the same medicine, but differences between biochemical base and pharmacological effect of these two velvet antlers have been observed. We compared the total polypeptide mapping of the two velvet antlers, discovering that nVAP is active polypeptide and only exists in the velvet antler of C. elaphus. sVAP is similar to nVAP in physicochemical property and biological activity. These studies extend the possible utility of sVAP to be the promising compound to prepare velvet antler polypeptide of C. elaphus.     

不同加工方式对鹿茸中水溶性多糖含量及单糖组成的影响

采用水提醇沉法提纯鹿茸中的水溶性多糖，苯酚-硫酸法测定其含量；多糖经水解、衍生后通过UPLC分析不同加工方式及不同部位鹿茸中单糖组成及含量的差异。结果表明，煮炸茸蜡片、粉片、纱片、骨片中水溶性多糖含量分别为1.74、1.67、1.03和1.13 g/kg，冻干茸为2.77、3.07、1.22和3.20 g/kg；排血茸4个部位水溶性多糖含量依次为1.55、1.78、0.96和0.77 g/kg，带血茸为1.69、1.64、1.01和1.31 g/kg。不同加工方式鹿茸中水溶性多糖单糖组成均检出甘露糖、氨基葡萄糖、核糖、葡萄糖醛酸、半乳糖醛酸、氨基半乳糖、葡萄糖和半乳糖8个组分，对于同一部位，煮炸茸单糖含量低于冻干茸，排血茸单糖（氨基葡萄糖和氨基半乳糖除外）含量低于带血茸；对于同一加工方式不同部位，表现出蜡片、粉片高于纱片、骨片。研究加工方式对鹿茸中水溶性多糖含量及单糖组成的影响，以期为鹿茸加工及其产品开发提供理论参考。     

鹿茸保健品中11种性激素的气相色谱-串联质谱法分析

建立了气相色谱-串联质谱法测定鹿茸保健品中11种性激素的分析方法。鹿茸中的性激素经固相萃取富集和净化,经七氟丁酸酐衍生处理。采用DB-5色谱柱(30 m×0.25 mm, 0.25 μm)、非线性梯度升温程序分离,在串联质谱多反应监测(MRM)模式下检测,外标法定量,实现了11种性激素的有效分离。11种性激素的检出限为1.0～5.0 μg/kg,线性相关系数为0.9916～0.9999,平均回收率为67.4%～99.1%,相对标准偏差为2.6%～13%。该方法准确,可靠,可满足鹿茸保健品中性激素含量的测定和确证。     

麇鹿茸样品的制备和化学成分分析

建立了麋鹿茸样品制备的工艺路线和方法,获得了易保存的干燥粉末样品,对麋鹿茸中的水分、粗蛋白、粗脂肪、膳食纤维、氨基酸、必需无机元素以及维生素的含量进行了测定,并与马鹿和梅花鹿茸进行了比较。结果表明,麋鹿茸与马鹿和梅花鹿茸的化学成分及含量相近,麋鹿茸中膳食纤维和必需无机元素含量高于其它两种鹿茸。     

Determination of Sex Hormones in Antler Velvet by High Performance Liquid Chromatography Tandem Mass Spectrometry

Eighteen sex hormones in antler velvet were determined by high performance liquid chromatography tandem mass spectrometry.The solid phase extraction was applied to eliminating the matrix effect.The exp...     

麋鹿茸、马鹿茸和梅花鹿茸营养成分的分析比较研究

对麋鹿茸,马鹿茸,梅花鹿茸中的水分,粗蛋白,粗脂肪,膳食纤维,水溶性和脂溶性维生素,氨基酸和无宏量及微量元素进行了测定,和比较,结果表明,麋鹿茸与马鹿茸和梅花鹿茸的化学分成及含量相近,鹿鹿茸中膳食纤维和必需无机元素含量高于其它两种鹿茸,填补了麋鹿研究的空白,为鹿资源的保护,发展,开发和利用提供了营养学依据。     

Rapid qualitative and quantitative evaluation of deer antler (Cervus elaphus) using near-infrared reflectance spectroscopy

Near-infrared (NIR) spectroscopy has been applied for both the qualitative and quantitative evaluation of the velvet deer antler. The most important parameters of determining the quality of velvet antler are the habitat (the country of origin) and ash content. Conventionally, the habitat is determined by examining the appearance of samples (by human eye), which lacks objectivity. Ash content is measured by an ignition method (measurement ash residue), however, it is too slow (4–5 h) to be used for rapid at-site measurement. Velvet antlers from three different habitats (China, New Zealand, and Russia), albeit the same species of Cervus elaphus, were evaluated in this paper. Soft independence modeling of class analogies (SIMCA) and partial least squares (PLS) were used for classification of habitat and determination of ash content. The habitat was successfully identified with over 80% accuracy, and the ash content prediction result using PLS regression showed good correlation with the reference ignition method with a standard error of prediction (SEP) of 1.264%.     

酶解鹿茸肽的制备、纯化及抗氧化活性

利用一种工业用碱性蛋白酶对新鲜梅花鹿鹿茸进行水解制备生物活性肽。首先,考察了酶用量、底物浓度和反应时间对水解反应的影响,确定了最佳水解条件为酶浓度1：150,底物浓度1：13,水解时间60 min。其次,利用硫酸胺分级沉淀法制备了鹿茸多肽粗提液,同时采用缓冲液保护其活性,再经Sephadex G-25凝胶层析柱进行分离纯化,得到了具有最强抗氧化活性的鹿茸肽组分（VAP-B）。最后,利用制备型高效液相色谱柱对VAP-B进一步纯化,得到了分子量在800以内的小肽活性组分（VAP-B1）,其蛋白含量为70.45%。在此基础上,对小肽活性组分VAP-B1进行了理化性质分析,检测其抗氧化活性,包括清除超氧阴离子,羟自由基以及抗脂质过氧化的能力和还原能力。实验结果表明,鹿茸肽VAP-B1浓度为20mg/ml时,对邻苯三酚自氧化的抑制率达到91.9%,有明显的清除超氧阴离子的作用;浓度为10mg/ml时,对羟基自由基的清除作用达到100%,且在一定的浓度范围内呈剂量依赖关系。此外,鹿茸肽VAP-B1具有一定的防止脂质过氧化和还原力作用。     

Effects of velvet antler with blood on bone in ovariectomized rats

In traditional Chinese medicine (TCM), both velvet antlers (VA) and VA blood can tonify qi, essence, and marrow, nourish the blood, and invigorate bones and tendons. In TCM, the combination of VA and VA blood is believed to have superior pharmacological effects. Scientific evidence supporting the traditional therapeutic preference for redder antler is needed. The effectiveness of the combination therapy of VA middle sections (VAMs) and VA blood (VAM-B) was first examined in promoting proliferation of mouse osteoblastic cells (MC3T3-E1). The anti-osteoporotic activity of VAM-B (ratio of VAM:VA blood = 1:0.2) was evaluated with ovariectomized (OVX) rats at a dose of 0.2 g/kg. In VAM-B-treated OVX rats, the body weight decreased 10.7%, and the strength of vertebrae and the femur respectively increased 18.1% and 15.4%, compared to the control. VAM-B treatment also recovered the estrogen-related loss of the right tibial trabecular bone microarchitecture. Alkaline phosphatase (ALP) significantly decreased, but estradiol did not significantly change in serum of VAM-B-treated OVX rats. We also provide an effective strategy to enhance the anti-osteoporotic activity of VAM. In conclusion, our results provide scientific evidence supporting the traditional therapeutic preference of redder antler and indicate that VAM-B is a potential therapeutic agent for managing osteoporosis.     

Antioxidant activity of protein hydrolysates from aqueous extract of velvet antler (Cervus elaphus) as influenced by molecular weight and enzymes

The crude protein hydrolysates from aqueous extract of velvet antler (AEVA) were prepared by simulated gastrointestinal digestion (SGI, pepsin-pancreatin) using pancreatin-pepsin, alcalase and neutrase. The resulting hydrolysates were separated by sequential ultrafiltration into four fractions. The antioxidant activities of peptide fractions were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging and Fe(2+)-chelating assays. Results showed that the hydrolysate prepared by SGI had a low degree of hydrolysis, which was significantly improved with altered proteases, such as pancreatin-pepsin and alcalase. Antioxidant activities of peptide fractions varied with molecular weight (MW) and the enzyme used. Generally, low-MW peptide fractions had higher ABTS radical scavenging activity and Fe(2+)-chelating ability, and high-MW peptide fractions were more effective in DPPH radical scavenging activity and reducing power.     

高效液相色谱–质谱法分析僵蚕蛋白酶解多肽

分析僵蚕蛋白酶解多肽类成分的相对分子质量和氨基酸组成。采用高效液相色谱–质谱联用正离子模式进行分析,以Hola C_(18)色谱柱(100 mm×2.1 mm,2.7μm)为分离色谱柱,以0.05%甲酸水溶液和0.05%甲酸–乙腈溶液为流动相,根据质谱一级、二级碎片离子信息,确定酶解多肽类相对分子质量信息和氨基酸组成。僵蚕样品经酶解后得到相对分子质量在500~1 000之间的多肽,经LC–MS分析,多肽由低于10个的氨基酸组成。高效液相色谱–质谱法分析平台可用于分析多肽化合物的相对分子质量和氨基酸组成,这有利于酶解多肽的生物活性分析。     

Exogenous Melatonin Activating Nuclear Factor E2-Related Factor 2 (Nrf2) Pathway via Melatonin Receptor to Reduce Oxidative Stress and Apoptosis in Antler Mesenchymal Stem Cells

Antler growth depends on the proliferation and differentiation of mesenchymal stem cells (MSCs), and this process may be adversely affected by oxidative stress. Melatonin (MLT) has antioxidant functions, but its role in Cervidae remains largely unknown. In this article, flow cytometry, reactive oxygen species (ROS) identification, qPCR, and other methods were used to investigate the protective mechanism of MLT in H₂O₂-induced oxidative stress of antler MSCs. The results showed that MLT significantly increases cell viability by relieving the oxidative stress of antler MSCs. MLT inhibits cell apoptosis by protecting mitochondrial function. We blocked the melatonin receptor with luzindole (Luz) and found that the receptor blockade significantly increases H₂O₂-induced hyperoxide levels and causes significant inhibition of mitochondrial function. MLT treatment activates the nuclear factor E2-related factor 2 (Nrf2) antioxidant signaling pathway, up-regulates the expression of NAD(P)H quinone oxidoreductase 1 (NQO1) and other genes and it could inhibit apoptosis. In contrast, the melatonin receptor blockade down-regulates the expression of Nrf2 pathway-related genes, but significantly up-regulates the expression of apoptotic genes. It was indicated that MLT activates the Nrf2 pathway through the melatonin receptor and alleviates H₂O₂-induced oxidative stress and apoptosis in antler MSCs. This study provides a theoretical basis for further studying the oxidative stress and antioxidant process of antler MSCs and, thereby, increasing antler yields.     

<em>In Vitro</em> Anti-diabetic Activities and Chemical Analysis of Polypeptide-k and Oil Isolated from Seeds of <em>Momordica charantia</em> (Bitter Gourd)

The amino acid and fatty acid composition of polypeptide k and oil isolated from the seeds of Momordica charantia was analysed. The analysis revealed polypeptide k contained 9 out of 11 essential amino acids, among a total of 18 types of amino acids. Glutamic acid, aspartic acid, arginine and glycine were the most abundant (17.08%, 9.71%, 9.50% and 8.90% of total amino acids, respectively). Fatty acid analysis showed unusually high amounts of C18-0 (stearic acid, 62.31% of total fatty acid). C18-1 (oleic acid) and C18-2 (linoleic acid) were the other major fatty acid detected (12.53% and 10.40%, respectively). The oil was devoid of the short fatty acids (C4-0 to C8-0). Polypeptide k and oil were also subjected to in vitro α-glucosidase and α-amylase inhibition assays. Both polypeptide k and seed oil showed potent inhibition of α-glucosidase enzyme (79.18% and 53.55% inhibition, respectively). α-Amylase was inhibited by 35.58% and 38.02%, respectively. Collectively, the in vitro assay strongly suggests that both polypeptide k and seed oil from Momordica charantia are potent potential hypoglycemic agents.     

鹿茸寡肽的制备及其促成骨细胞的增殖作用

从梅花鹿鹿茸中提取了天然鹿茸总多肽(VATP). 在进一步分离纯化的过程中, 筛选出具有促进成骨细胞增殖的高活性肽组分(VAP-B), 并通过制备型HPLC对其纯化, 得到了分子量分布约为200~600的小肽活性组分(VAP-B2). 细胞周期分析结果表明, 鹿茸肽VAP-B2组分促进了人成骨肉瘤细胞OS-732的周期转化, 使其细胞周期S期细胞指数明显增加, 即表现为S期DNA含量明显提高. 碱性磷酸酶(ALP)活性检测结果表明, 随着鹿茸肽VAP-B2剂量的增加, ALP的活性明显增加. 这与成骨细胞增殖分化及成熟过程中, 细胞的周期性变化和骨形成标志物碱性磷酸酶水平的明显增高相符.     

A new monitoring system of cultured myocardial cell motion: effect of pilose antler extract and cardioactive agents on spontaneous beating of myocardial cell sheets

Effects of various cardioactive agents and a water extract of the pilose antler of Cervus nippon var. mantchuricus on periodic beating of cultured myocardial cell sheets were examined by using an image analyzing system. Norepinephrine increased the beating rate and the beating amplitude, whereas digoxin and forskolin enlarged only the beating amplitude. Verapamil and propranolol decreased both the beating rate and the beating amplitude. The water extract of the pilose antler showed no remarkable effects in a standard medium (2.1 mM Ca2+). However, it significantly increased the beating amplitude when the beating was suppressed by replacement with a low calcium medium (0.5 mM Ca2+). A similar effect was found for 70% ethanol-soluble and -insoluble fractions of the extract.     

Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.     

Analysis by two-dimensional electrophoresis of the effect of salt stress on the polypeptide patterns in roots of a salt-tolerant and a salt-sensitive cultivar of wheat

The effect of salt stress on the polypeptide levels in roots of two wheat (Triticum durum) cultivars with different sensitivity to NaCl (cv. Ben Bachir, sensitive; cv. Chili, tolerant), was examined by two-dimensional polyacrylamide gel electrophoresis. Blue-stained gels were analyzed by visual inspection to identify changes that resulted when seedlings were grown in the presence of 200 mM NaCl for four days. Although the protein patterns for control and salt-stressed seedlings were qualitatively similar, the net synthesis of a 26 kDa polypeptide was significantly changed. This observation was mainly noticeable in the more tolerant cultivar. With the intention of identifying its function, the NH2-terminal of this polypeptide was sequenced. A 20 amino acid sequence was obtained and compared to sequences available in different databases. Possible roles of this polypeptide, depending on the homologies of its amino acid sequence with known proteins, in salinity tolerance are discussed.     

Ionic strength-dependent pK shift in the helix-coil transition of grafted poly(L-glutamic acid) layers analyzed by electrokinetic and ellipsometric measurements

Surface-bound layers of poly(L-glutamic acid) prepared by a recently described "grafting-from" method were analyzed with respect to electrical charging and structural alterations upon variation of pH and concentration of the background electrolyte in aqueous solutions. The microslit electrokinetic setup (MES) was utilized for the combined determination of zeta potential and surface conductivity on the basis of streaming potential and streaming current measurements at polypeptide layers in contact with aqueous electrolyte solutions of varied composition. In situ ellipsometry was applied at similar samples immersed in identical aqueous solutions to investigate the influence of the solution pH on the structure of the polypeptide layers. Zeta potential and Dukhin number versus pH plots revealed the dissociation behavior of the surface-bound polypeptides indicating a significant shift of the pK of their acidic side chains correlating with the concentration of the background electrolyte potassium chloride and the related variation of the Debye screening length. Surface conductivity data pointed at a more expanded structure of the polypeptide layer in the fully dissociated state as an increased ion conductance in this part of the interface was determined. The occurrence of a strong increase of the thickness and a corresponding decrease of the refractive index for the coil state of the layer strongly supports the findings of the electrokinetic measurements. This fully reversible "switching" of the layer structure was attributed to helix-coil transitions within the grafted polypeptides induced by the dissociation of carboxylic acid functions of the polypeptide side chains. The shift of the "switching pH" of the surface-bound poly(L-glutamic acid) layers at varied concentrations of the background electrolyte was interpreted as a result of the pK shift of the carboxylic acid groups of the polypeptide side chains. The observed patterns prove that the electrostatic interactions causing this shift occur within but not between the grafted chains.