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题名: 维吾尔药罗勒子化学成分及其生物活性研究
作者: 胡尔西丹.伊麻木
答辩日期: 2013-11-19
导师: 阿吉艾克拜尔·艾萨、热娜·卡斯木
专业: 有机化学
授予单位: 中国科学院大学
授予地点: 北京
学位: 博士
关键词: 罗勒子多糖 ; HPTLC-MS ; 抗氧化 ; 抑制蛋白酪氨酸磷酸酯酶1B(PTP1B) ; 结构特征研究
摘要: 罗勒(Ocimum basilicum L.),为唇形科罗勒属植物中的一种,在我国包括栽培在内共5 种,3 个变种。罗勒子为罗勒的干燥成熟果实,记载于中华人民共和国卫生药品标准 “维吾尔药分册”,是维吾尔族民间常用药材之一。大量文献显示,目前为止,前人对罗勒子的研究主要集中在对种子的培育、生长过程的影响因素及其杀虫作用等方面,虽然印度科研人员对罗勒子粘液质的多糖进行了酸性多糖的分离和单糖组成分析,但由于研究年限过早、研究手段简单,单糖组成及其含量分析并不全面,而且对其脂肪酸、挥发油和其它化学成分及生物活性等方面的研究还相当欠缺,国内外研究基本空白,其活性成分和作用机理尚不明确,药材指标成分的选择缺乏科学依据,造成其质量标准低,亟待提高。为了寻找其有效成分和科学食用方法,揭示其抗氧化和降血糖等作用的物质基础,本论文以新疆罗勒子为研究对象,采用抗氧化、抑制蛋白酪氨酸磷酸酯酶1B(PTP1B)等活性筛选为导向的研究手段,对新疆罗勒子中水溶性、碱溶性和酸溶性多糖进行了系统的提取分离和纯化研究,结合阴离子交换树脂、分子排阻色谱等现代分离分析技术,对新疆和田产罗勒子酸性多糖成分进行进一步的分离纯化和结构特征研究,采用高效薄层色谱-质谱连用技术、毛细管电泳等技术分析多糖提取物单糖组成,并考察了酸性多糖的抗氧化和降糖生物活性,同时建立了罗勒子脂肪酸成分的超临界二氧化碳提取工艺,为罗勒子资源的综合利用提供了科学依据,主要研究内容如下:1. 罗勒子脂肪酸成分提取工艺研究及其蛋白酪氨酸磷酸酯酶1B(PTP1B)抑制作用本研究通过索氏提取、微波提取和超临界二氧化碳提取等现代提取工艺技术,对罗勒子的脂肪酸成分进行了系统的提取工艺研究,确定了最佳提取工艺,同时利用气相色谱质谱(GC/MS)仪鉴定出脂肪酸的主要化学成分的类型,并评价其食用价值和体外蛋白酪氨酸磷酸酯酶1B(PTP1B)抑制作用;研究结果证实,索氏提取方法罗勒子出油率为14 %,其中α-亚麻酸为62.88%、亚油酸为20.66 %、棕榈酸为10.67 %、硬脂酸为5.79 % 为四个主要脂肪酸组成成分;超临界二氧化碳提取脂肪酸最佳工艺为,提取压力300 bar,提取温度40°C,提取时间4 小时,在此工艺条件下提取的罗勒子油颜色明亮清晰品质好,出油率可达17.05 %,其中不饱和脂肪酸成分含量达到85 %;微波协助提取方法罗勒子脂肪酸的最佳提取工艺为:提取温度为40 °C,微波功率为400 w,提取时间为6 分钟,此方法罗勒子出油率为25.8 % 其中不饱和脂肪酸含量达到88.41 %;研究证实,罗勒子油脂的酸值为41.97,碘值为36.26,皂化值为156.17,各参数均在中华人民共和国药典指定的范围内,因此证明罗勒子油脂可作为食用油;对罗勒子脂肪酸进行PTP1B 的抑制作用研究,结果表明脂肪酸对PTP1B 有较强的抑制作用,其IC50 为11.12 μg/mL。2.罗勒子挥发油成分及抗氧化活性研究通过水蒸气蒸馏法提取了新鲜采集罗勒子中的挥发油,并采用GC-MS 仪器鉴定出以亚麻油酸(13.83 %)、正棕榈酸(13.80 %)、长叶薄荷酮(8.31 %)、反式斯巴醇(6.72 %)等四个化合物为主的60 种化学成分;新疆罗勒子挥发油与不同产地罗勒子挥发油成分对比结果显示,新疆罗勒子挥发油化合物类型以酸、酮、醇等为主;而且由于罗勒的产地、采收季节、提取方法、甚至储藏等条件不同,所提取的罗勒挥发油的主要成分也不同。通过抗氧化活性测定(清除DPPH 自由基能力)证明了罗勒子挥发油具有较好的清除DPPH 自由基的作用,其抑制率与浓度成正比。3.罗勒子多糖的提取分离、制备及其单糖组成研究在这项研究中,连续提取和纯化方法制备了水溶性多糖(WSPS-C)、热水溶性多糖(WSPS-H)、半纤维素(HC)、果胶(PS)、热水溶性酸性多糖(WSPS-H-A)和半纤维素酸性多糖(HC-A)等6 种多糖提取物,对其理化性质分析结果表明,罗勒子粗多糖提取物都是糖蛋白缀合物,经测定,其中WSPS-C 中性糖含量为76.5 %,糖醛酸含量为9.3 %,蛋白质含量为8.0 %;WSPS-H 中性糖含量为67.3%,糖醛酸含量为21.3 %,蛋白质含量为9.0 %;HC 中性糖含量为43.4 %,糖醛酸含量为16.2 %,蛋白质含量为17.0 %;PS 中性糖含量为43.2%,糖醛酸含量为41.3 %,蛋白质含量为1.4 %;它们至少含有9.3 %的糖醛酸成分。利用高效薄层层析技术(HPTLC),以乙酸异丙酯-醋酸乙酯-甲醇-水5:4:1:0.1(v/v/v/v)为展开剂,并在370 或630 nm 光密度定量测定方法,对罗勒子6 种多糖提取物进行其单糖成分的定性和定量分析。结果显示,罗勒子多糖主要由果糖(HRF80),葡糖醛酸(HRF 58),半乳糖醛酸(HRF51),鼠李糖(HRF 40),木糖(HRF 25),阿拉伯糖(HRF 18)和半乳糖(HRF 9)等7 种单糖组成,其中木糖、葡萄糖醛酸和果糖为主要组成成分,它们在总多糖的百分含量分别为45 %、31 % 和21 %。所有的提取物含有糖醛酸,其范围在3-24 %;研究中在葡糖醛酸上面发现一个未知的化合物,并采用HPTLC-UV/Vis-MS 技术,确定其为葡萄糖醛酸异构体。同时,建立了柱前衍生-高效毛细管电泳分析罗勒子水溶性总多糖的方法,并测定了罗勒子水溶性多糖中的葡萄糖、半乳糖醛酸、鼠李糖、甘露糖、阿拉伯糖、葡糖醛酸和半乳糖等7 种单糖组成的含量,研究结果表明,其单糖组成的摩尔比分别为3.54: 2.56: 1.56: 0.46: 0.21:0.99:0.12 。生物活性筛选结果表明,罗勒子总多糖提取物WSPS-C、 WSPS-H、 PS 和 HC 都具一定的降糖活性,它们体外具有较强的抑制PTP1B 活性,它们的IC50 值为分别为 8.2, 2.2, 70.9 和0.8μg/mL, 其中半纤维素总多糖的降糖活性为最强IC50=0.8 μg/mL。通过进一步文献查阅和结合单糖组成研究结果,推断罗勒子酸性多糖的连接键型;推断罗勒子多糖含有(1→4)链接的木聚糖主链携带的分支点,在C-2 和C-3 的木糖残基,并显示的侧链的结构特征。4. 罗勒子酸性单体多糖的分离、纯化、制备及其结构特征研究采用离子交换树脂、凝胶渗透和分子排阻色谱(Sephadex G-100)等分离纯化技术对罗勒子热水溶酸性级分多糖部位(WSPS-H-A),进行其单体多糖成分的分离、纯化和制备,并首次从罗勒子中分离得到了LLZ-Hot-200-0.2-15.2-G-I、LLZ-Hot-200-0.2-15.2-G-II 、LLZ-Hot-200-0.2-23-G 、LLZ-Hot-200-0.2-32.6-G 、LLZ-Hot-200-0.2-25.6-G ; LLZ-Hot-200-0.5-I-G-I 、LLZ-Hot-200-0.5-I-G-II 、LLZ-Hot-200-0.5-II、LLZ-Hot-200-0.5-III;LLZ-Hot-200-1.0-I、LLZ-Hot-200-1.0-II等15 个单体酸性多糖成分,其中9 个单体化合物结构用核磁共振(NMR)、红外(IR)、紫外(UV)和LC-MS 的等光谱技术进行了分析,并总结了罗勒子多糖的结构特征。采用紫外和红外光谱分析考察其结构表明,罗勒子酸性单体多糖都是非糖蛋白缀合物(多糖仪没有紫外吸收);从罗勒子的中红外光谱结构分析看出,醇羟基的基频是在3409-3437cm-1 左右,峰形尖锐的吸收峰是 OH 的伸缩振动吸收峰;2923 cm-1 处的吸收峰是C-H 的伸缩振动吸收峰,1645cm-1 处的吸收峰是OH 的弯曲振动吸收峰;核磁共振氢谱1H NMR 分析结果显示,糖环碳上大部分质子的化学位移由于受羟基的屏蔽作用而位于3~4 ppm 范围内,信号重叠严重,解析困难; 核磁共振碳谱13C NMR 分析结果显示,罗勒子酸性多糖的化学位移范围较1H NMR 广,达200 ppm,具有较好的分辨率。
英文摘要:
Study on chemical compositions and biological activity of Fruits of Ocimum Basilicum from Xinjiang of China Ocimum basilicum L, also known as Nine-layer Tower, Saint Joseph’s Wort and Sweet Basil, is a dwarf and tender culinary herb in genus Ocimum of family Lamiaceae. Fructus Ocimum Basilicum is the dry mature fruit of Ocimum basilica. As recorded in the ancient China medical book Compendium of Materia Medica, Fructus Ocimum Basilicum is rich in lots of mineral elements, vitamins, carotene, and amino acids and so on, which has the functions of improving eyesight, nourishing the kidney, eliminating constipation and fitness. Fructus Ocimum Basilicum is cold in nature and could stop diarrheic, dispel heat from blood to stop bleeding, reduce fever and peace the heart,it is mainly used to cure damp-heat or blood diseases, such as damp-heat dysentery, bellyache, diarrhoea, hematochezia, hemorrhoid hemorrhage, febrile heart disease and so on. Modern research finds out that Fructus Ocimum Basilicum has the functions of diuresis, antioxidation, antisepticise, anti-inflammation, bacteriostasis, curing angiocardiopathy and so on. The present study mainly focus on systemically study of chemical compositions that essential oil, fatty acids as well as polysaccharides compositions of seed of Ocimum Basilicum that caltivated in Xinjiang of china, while the reseach on the chemical components as well as biologically study of Fructus Ocimum Basilicum L. from Xinjiang are rarely reported. 1. Study on Extraction Technology of lipids from the seed of Ocimum basilicum L by Supercritical CO2 methods The studies on the optimum conditions for the extraction of Ocimum basilicum L seeds oil by supercritical fluid were carried out. The primary and secondary effect factors on the extraction of Ocimum basilicum seeds oil by supercritical fluid were as followed: pressure > flow quantity > temperature >. The optimum conditions were: extraction time 4 hours, extraction temperature 40 °C, extraction pressure 300 MPa and flow quantity 6L/ h. The yield seed of Ocimum basilicum L oil was 17.1 %. 2. Study on the Chemical Components and Antioxidant Activity of Essential 0il from Ocimum basilicum Seeds. Volatile oil of Ocimum basilicum L seeds was extracted by stem distillation methods and contents of it was determned by GC-MS analysis. The results showed that 65 components were identified and they are account for 96.59 % of the total mass volatile oil. The dominant compositions of essential oil of Ocimum basilicure seeds were Linoleic (13.83 %), n-Hexadecanoic acid (13.80 %), Pulegone(8.31 %), Spathulenol (6.72 %). 3. Chemical Components and PTP1B inhibition activity of fatty acid from seed of Ocimum basilicum L. In this paper, a fatty acid composition seed of Ocimum basilicum L was extracted by Soxhlet extractor and chemical composition of it was quantified by GC/MS after methyl esterification of fatty acids. The results indicated that Fatty acid yield was 14.05 %, and the four main fatty acids were identified, they are are linoleic acid, hexadecanoic acid, α-linolenic acid and octadecanoic acid. The content of unsaturated fatty acids up to 83.54 %, among them, the α-linolenic acid was 62.88 %, the linoleic acid was 20.66 %, hexadecanoic acid was 10.67 %, and the octadecanoic acid was 5.79 % respectively. The fatty acids had the strong inhibiting effect on PTP1B, and its IC50 value was 11.12 μg/ mL. 4. Study on microwave assistant extraction technology of fatty acid composition seed of Ocimum basilicum L and evaluation of its food value. As a seed of plant its oil contents can be imagined, but the fatty acid composition of seed of basil have not been reported yet, in order to provide a scientific basis for the comprehensive use of basil in the future, this study we established microwave assisted extraction technology for fatty acid composition of basil seeds by a single factor method, and gained fatty acids yield with 25.8 % and analyzed its compositions by GC/MS; research results show that the optimum extraction conditions are: temperature at 40 °C, microwave power 400 w, extraction time 6 minutes; extraction contain 88.41 % unsaturated fatty acids, thay are the linoleic acid 69.73 %, linoleic acid 18.68 %, the palmitic acid 6.85 % and the stearic acid 4.74%. Ocimum basilicum L oil has an acid value of 41.97, an iodine value of 36.26 and a saponification value of 156.17,those parameters are within the range of specified in the Pharmacopoeia of the People's Republic of China, therefore oil of seeds of Ocimum basilicum L can be used as edible oil. 5. Quantification of Anti-diabetic Polysaccharides in Seeds of Ocimum basilicum by High Performance Thin Layer Chromatography (HPTLC). In this study, six polysaccharide fractions (designated WSPS-C, WSPS-H, PS, HC, WSPS-H-A and HC-A) were isolated from the seeds of Ocimum basilicum by the sequential use of water, boiling water, diluted acid and base extraction respectively, ethanol precipitation and ion-exchange chromatography. High-performance thin-layer chromatography (HPTLC) analysis was performed sμgar detected and quantified on glass silica gel 60 F254 plates developed with 10 mL i-propylacetate – ethylacetate – methanol–water 5:4:1:0.1 (v/v/v/v); migration distance 60 mm; drying time 30 s before and 2 min after development. Densitometric quantification was performed at λ = 630 nm and λ= 370 nm (the wavelength of maximum absorption of monosaccharide) by fluorescence scanning using a CAMAG TLC Scanner 3. The results revealed that seeds of Ocimum basilicum polysaccharides consisted primarily of Fructose,Glucuronic acid , Galactonic acid , Rhamnose, Xylose, Arabinose and Galactose. The Xylose, Glucuronic acid,Fructose and Arabinose are major compositions, they account for 35.2 %, 33.8 %, 24.7 % and 23.7 % respectively seeds polysaccharides of Ocimum basilicum L. Moreover, all fractions contained at least 5.3% uronic acid. In addition, fractions WSPS-C, WSPS-H, PS and HC had significantly higher inhibitor activity of protein tyrosine phosphatetase-1B (PTP1B) in vitro with IC50 value 24.5, 8.2, 70.9 and 0.76 μg/mL respectively. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of seeds of Ocimum basilicum extracts, and sμggest the possibility of using seeds of Ocimum basilicum polysaccharides as an antidiabetic adjuvant. 6. Extraction of an antidiabetic polysacchrides from seeds of Ocimum basilicum and determination of the monosaccharide composition by precolumn high-efficiency capillary epectrophoresis. A water-soluble polysaccharides (WSPS) extracted from seeds of O. basilicum, and monosaccharide composition was determination by precolumn high-efficiency capillary electrophoresis, and in vitro screening for antidiabetic activity. The results showed that WSPS from seeds of O. basilicum consisted of glucose, galacturonic acid, rhamnose, mannose, arabinose, glucuronic acid, and galactose in mole ratio 3.54: 2.56: 1.56: 0.46: 0.21: 0.99: 0.12, respectively. The antidiabetic activity of WSPS from seeds of O. basilicum was determined by measuring the inhibitory activity for protein tyrosine phosphatase 1B in vitro. The inhibitory activity for protein tyrosine phosphatase 1B was IC50 = 8.20 mg/mL. 7. Isolationg、purification of individual polysaccharides from the water solible (hot water) acidic polysachride fration and characterization their structure. We have isolated 15 individual acidic polysachchrides from WSPS-H-A fraction with use of ion-exchange resins, gel permeation chromatography and size exclusion chromatography with Sephadex G-100 for the frst time and among the 9 polysaccharides stucuture were characterized by H1-NMR, C13-NMR、IR、UV、and LC-MS technich.
内容类型: 学位论文
URI标识: http://ir.xjipc.cas.cn/handle/365002/3396
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胡尔西丹.伊麻木. 维吾尔药罗勒子化学成分及其生物活性研究[D]. 北京. 中国科学院大学. 2013.
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