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新疆四种动物骨髓蛋白与油脂的分离分析及其应用研究
帕尔哈提·柔孜
Thesis Advisor阿布力米提·伊力
2018-06-02
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Name博士
Degree Discipline有机化学
Keyword动物骨髓 蛋白与多肽 液质联用色谱 营养成分 生物活性
Abstract

动物类中药是中国传统药学的重要组成部分,其中动物骨骼和骨髓具有悠久的药用历史,记载于《本草纲目》、《中药大辞典》、《白地依药书》等。新疆作为畜牧业大省,动物骨髓来源丰富,疆内医院使用动物骨髓为原料的制剂由来已久,具有祛风止痛、消炎退肿、软筋松肌等作用,治疗骨折僵硬、关节炎、风湿等疾病药效较好。但现有制备方法陈旧,有效部位产率低,活性弱,特征成分至今尚未明确,质量控制不规范。因此,本文将新疆地产羊、牛、马和骆驼腿骨骨髓为研究对象,对其中的蛋白、多肽、氨基酸、油脂与微量元素等成分展开系统的分离分析及活性研究,为动物骨髓资源的有效利用提供科学依据。一、骨髓蛋白和多肽的制备与鉴定1)牛骨髓蛋白提取方法的筛选:以牛骨髓蛋白含量、提取率和抗菌活性作为指标,对10种不同提取方法进行了比较研究。结果显示,水、0.5 mol/L NaCl和Tris-HCl溶液为理想提取溶剂,其蛋白含量分别为52.20 ± 0.58 mg/mL,50.19 ± 0.21 mg/mL,43.21 ± 0.05 mg/mL。进一步LC/MS分析得出,0.5 mol/L NaCl提取部位主峰约含有33种多肽段,带电荷的多肽数量多于其他部位,适合于进一步分离分析。2)牛骨髓蛋白提取方法优化及超滤膜富集工艺研究:通过单因素和正交试验优化了NaCl提取工艺,最佳提取条件为:NaCl浓度0.5 mol/L,提取次数2次,提取温度45℃,提取时间3 h,提取率为55.3%,蛋白含量达56.7 mg/mL。采用响应面法优化超滤膜富集工艺,最佳参数为:10 kDa载留膜,操作压力0.2 MPa,浓缩倍数5倍,温度40 ℃,膜通量达25.29 L /(m2.h)。该方法得到的蛋白质含量提高至76.9 mg/mL。3)牛骨髓多肽肽段的鉴定:选用上述方法制备的牛骨髓水提蛋白、0.5 mol/L NaCl提取液超滤膜分离部位和葡聚糖凝胶纯化部位,通过二维电泳技术进行分离,并用MALDI-TOF-TOF等技术确定多肽段分子量和氨基酸序列。共鉴定20种具有活性多肽段,其中抗氧化肽10种,抗菌肽7种,降压肽3种。4)三种骨髓蛋白的提取与分离分析:以上述研究基础上,采用上述最佳三种提取方法对骨髓蛋白进行制备,筛选最佳提取部位,并进行抗菌、抗氧化活性筛选。结果显示,水提法蛋白提取率为:马骨髓>羊骨髓>骆驼骨髓;硫酸铵分级沉淀法中30%马骨髓部位蛋白提取率最高。0.5 mol/L NaCl提和膜分离中,骆驼骨髓蛋白10 kDa载留部位含量最高(72.63 mg/mL)。FT-IR分析显示3种骨髓水提蛋白二级结构以α-螺旋为主。SEM分析表明三种骨髓水提蛋白均有特定的表面结构。抗氧化结果表明,水提物中马和牛骨髓蛋白抗氧化能力较高,IC50值分别为0.573和0.834 mg/mL。二、 四种动物骨髓营养成分评价1)四种动物骨髓氨基酸组成分析:分别制备骨髓粉末和骨髓蛋白并评价水解氨基酸组成。结果显示均含有17种氨基酸,骨髓蛋白氨基酸种类比较齐全,药用氨基酸含量变幅为158.93-293.84 mg/g。氨基酸总量排序为:马骨髓蛋白>牛骨髓蛋白>羊骨髓蛋白>骆驼骨髓蛋白。马骨髓蛋白中人体必需氨基酸/氨基酸总量比例较接近于世界粮农组织/世界卫生组织(FAO/WHO)提出的理想氨基酸标准。2)四种动物骨髓常微量元素分析:分别测定骨髓粉末和骨髓蛋白中常微量元素含量。结果得出,骨髓蛋白中常微量元素含量高于骨髓粉末;四种骨髓蛋白中Na、K、Ca、P和Mg等5种常量元素的总含量排序为:马骨髓蛋白>骆驼骨髓蛋白>牛骨髓蛋白>羊骨髓蛋白。马骨髓蛋白中必须微量元素含量最丰富,其中Ca、P含量分别达24.92 mg/g、10.83 mg/g。3)四种动物骨髓油不饱和脂肪酸的富集与活性比较:采用四种溶剂(正己烷、石油醚、无水甲醇和无水乙醇)对牛骨髓油进行提取、分析。结果表明,正己烷提取效果最好,饱和脂肪酸/不饱和脂肪酸(SFA/UFA)比例为1/1.41。四种骨髓粉末经正己烷提取后,SFA/UFA比例分别为1/1.417、1/1.327、1/1.912、1.285/1;经尿素包合后,SFA/UFA分别提高至1/1.518、 1/1.390、 1/2.037、 1.216/1;经冷冻结晶后,SFA/UFA分别提高至1/1.920、1/2.141、1/2.360、1/1.157。冷冻结晶法能有效地富集不饱和脂肪酸,富集后抗菌和抗氧化活性均增强。三、马骨髓复方制剂成分与初步药理作用研究1)选用马骨髓为基质,制备复方制剂,测定其油脂、多糖、蛋白、常微量元素含量,分析其改善新西兰大白兔左膝关节挛缩模型的药理活性。结果表明,马骨髓复方含油率60-66%,SFA/UFA比例为1/1.71,其中不饱和脂肪酸含量达63.14%。抗氧化活性结果显示,马骨髓复方清除自由基能力较强。2)建立新西兰大白兔左膝关节挛缩模型,将复方药涂抹4周后,与原药相比,复方药组关节活动度(Range of Motion)、股动脉收缩期峰值血流速度(Peak systolic blood flow velocity,PS)和舒张末期血流速度(End diastolic blood flow velocity,ED)均有统计学差异(P<0.05)。马骨髓复方改善关节挛缩模型效果比原药好。

Other Abstract

Animal origin medicine is an important part of traditional Chinese medicine,recorded in the traditional ethnic medicine with local characteristics. Xinjiang is the largest livestock producing region in China. But, there are few reports concentrated on the research about chemical composition of animal bone, especially bone marrow. Bone marrow is rich in proteins, oils and trace elements, there are a great number of biological activities, including anti-inflammatory, treat osteoarthropathy, activating blood circulation and relieve pain.Therefore, the research about animal bone marrow has broad application prospects. This dissertation focused on the separation and analysis of the proteins, peptides, amino acids, oils and trace elements of the bone marrow of Xinjiang characteristic animal, like camels, cattle, horse and sheep, then study the biological activity of the above mentioned compounds. The study aimed to provide technical support for the full utilization and scientific application of bone marrow.1. Preparation of bone marrow proteins(BMP) and peptides1)Screening methods for bovine bone marrow protein(BBMP) extraction and selection of effective parts:Protein content, yield and antimicrobial activity as a index, compared effect of 10 different extract methods.The results showed that water, NaCl, Tris-HCl solution as perferable solvent. The protein content were 52.20 ± 0.58mg/mL,50.19 ± 0.21 mg/mL,39.27 ± 0.25 mg/mL, respectively. Through LC-MS investigation determined 34 kinds of peptides in the extract of 0.5 mol/L NaCl fraction, in which majority consisted of charged polypeptides. 2)NaCl salt extraction were optimized using single factor test and orthogonal test, optimun condition were: concentration 0.5 mol/L, extraction tempereture 45℃, extraction time 3 h, extraction times 2, maximum protein content and yield were 56.7 mg/mL and 55.3%. Utrafiltration membrane separation process were optimized, optimun condition were: select 10 kDa membrane, operation pressure 0.20 MPa, concentration rates 5, extraction tempereture 40℃, maximum membrane flux 25.29 L /(m2.h) .3) Sequences of polypeptides from bovine bone marrow were determined using 2D electrophoresis and MALDI-TOF-TOF method. 20 types of polypeptides were separated, 10 of them were anti-oxidative, 7 of them were antibacterial and 3 of them were hypotensive polypeptide. The result was in accordance with previous reports.4) Extraction and separation of BMP from three types of animals (sheep, horse and camel): On the basis of BBMP extraction result, the other three animal BMP were extracted by using above mentioned three perferable solvent, antimicrobial and antioxidant activity were evaluated, structures were characterized by SEM and FT-IR. Results showed that, content of proteins extracted by water were decreased in the order of horse bone marrow protein(HBMP) > sheep bone marrow protein(SBMP) > camel bone marrow protein(CBMP). 10 kDa penetration part of CBMP salt extraction have the highest content(72.63 mg/mL). All of the four kinds of BMP have amide A, B, I, II, III band characteristic absorption peaks in FT-IR spectra. The secondary structure of protein mainly consists of the α- helix. SEM micrographs revealed that all of the proteins have characteristic morphological structures. The DPPH radical scavenging ability of HBMP and BBMP was higher than another two types BMP, IC50 value were 0.573 mg/mL, 0.834 mg/mL, respectively.2. Nutritional evaluation of bone marrow from four species animal 1) Amino acid composition of four species animal bone marrow. Prepare bone marrow powder (BMPD) and BMP,then determine amino acid composition. The results showed that these extracts contained 17 kinds of amino acid and there have full range of amino acid in BMP. Total amnio acid order was: HBMP> BBMP> SBMP> CBMP. Among them HBMP essential amino acid/total amino acid (E/T) and essential amino/non-essential amino acid (E/N) ratios were close to food and agriculture organization /world health organization (FAO/WHO) ideal amino acid pattern report.2) Analysis of the trace elements content in four species animal bone marrow: It was found that, a large number of trace elements are condensed in BMP, beneficial trace elements of HBMP was the most abundant, of which the content of Ca and P was as high as 24.92 and 10.83 mg/g. 3) Preparation of oil and unsaturated fatty acids from four species animal bone marrow. Four kinds of solvents (n-hexane, petroleum ether, anhydrous methanol and absolute ethanol) were used to extract bone marrow oil(BMO). The results showed that, n-hexane was the optimum solvent, ratios of saturated fatty acid/ unsaturated fatty acids(SFA/UFA) were 1/1.41. The ratio of SFA/UFA in n-hexane extracts were 1/1.417, 1/1.327, 1/2.140 and 1.285/1 for sheep, bovine, horse and camel BMO. SFA/UFA ratio of urea inclusion method was 1/1.518, 1/1.390, 1/2.037 and 1.216/1, respectively. SFA/UFA ratio of frozen-crystallization method in acetone was 1/1.920, 1/2.141, 1/2.360 and 1/1.157,respectively. Among them, acetone crystallization method has great effect on enrichment of unsaturated fatty acid. All four kinds of BMO have antioxidant activity.3. Study on the main components and pharmacological effects of medicinal preparation. Select HBM to prepare compound medicine, and analyze the oil, protein, polysaccharide,trace element content. Newzealand rabbit left knee joint contracture model was established to explore the curative effect of compound medicine. The results showed that active component for preliminary identification was cycloartenol. After 4 weeks treatment, the related knee range of motion (ROM), peak systolic blood flow velocity (PS) and end diastolic blood flow velocity (ED) was measured.Compare with the control group (hospital preperation), the value of ROM, PS and ED of treated group (compound medicine) have statistical difference (P<0.05). In conclusion, the compound medicine prepared on the base of HBM has promising treatment properties.

Document Type学位论文
Identifierhttp://ir.xjipc.cas.cn/handle/365002/5452
Collection资源化学研究室
Recommended Citation
GB/T 7714
帕尔哈提·柔孜. 新疆四种动物骨髓蛋白与油脂的分离分析及其应用研究[D]. 北京. 中国科学院大学,2018.
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