藏药材波棱瓜子化学成分与保肝作用研究

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	藏药材波棱瓜子化学成分与保肝作用研究
其他题名	Study on Chemical Components and Liver-protective Effects of Tibetan Medicine Herpetospermum Seed
	赵先恩
学位类型	博士
导师	索有瑞
	2009-05-27
学位授予单位	中国科学院西北高原生物研究所
学位授予地点	西北高原生物研究所
关键词	波棱瓜子超临界co2萃取种子油脂肪酸氨基酸单糖黄酮保护化学性肝损伤
摘要	波棱瓜子(Herpetospermum seed)为葫芦科波棱瓜属波棱瓜Herpetospermum pedunculosum (Ser.) Baill (H. Caudigerum Wall) 的干燥成熟种子，为藏医常用药，产于中国西藏、青海、四川、云南等地和印度、尼泊尔等国。藏医认为其能泻肝火胆热、解毒、助消化，用于治疗赤巴病、肝病及消化不良。波棱瓜子是藏药中治疗肝病的首选药物之一，在用于治疗肝病的藏药处方中，绝大多数有波棱瓜子配伍。波棱瓜子中脂肪油含量高，其中油酸、亚油酸、亚麻酸等不饱和脂肪酸含量较高，资料表明，不饱和脂肪酸具有保护化学性肝损伤、抗氧化、预防肿瘤、提高免疫、预防心脏病和高血压等多种生理功能，具有良好的开发利用前景。对波棱瓜子进行单味药的研究，采用现代工艺技术开发具有保肝作用的新药，既符合藏药现代化的发展要求和思路，又有利于波棱瓜子相关药物的推广和使用。本学位论文以波棱瓜子的化学成分和保肝作用为主要研究内容，探索建立了脂肪酸、氨基酸、单糖的高灵敏柱前衍生HPLC-MS分析方法和黄酮化合物的HPLC-DAD-MS分析方法，并对波棱瓜子油的保护急性化学性肝损伤作用进行了动物实验药效学研究，取得了以下研究结论。 1、采用超临界CO2萃取技术萃取波棱瓜子油，考察了萃取温度、压力、CO2流量、时间以及波棱瓜子粉碎粒径对种子油萃取率的影响，确定了最佳萃取工艺参数为：萃取温度50℃，萃取压力35 MPa，CO2流量35 L/h，粒径80目，萃取时间3.5 h。 2、以1-[2-(对甲苯磺酸酯)乙基]-2-苯基咪唑[4,5-f] 9,10-菲(TSPP)作为柱前衍生试剂，建立了39种脂肪酸(30种饱和与9种重要的不饱和脂肪酸)的高效液相色谱荧光检测定量分析和在线质谱快速定性的分析方法(HPLC-FLD-MS)，分析对比了超临界CO2萃取、超声波萃取、微波回流萃取的种子油脂肪酸含量差异。同时，对比了酸催化与碱催化的甲酯化法在GC-MS分析波棱瓜子油脂肪酸时的优劣，并讨论了液相色谱与气质联用分析脂肪酸的特点。 3、以2-(11H-苯[a]咔唑)乙基氯甲酸酯(BCEC-Cl)作为柱前衍生试剂，建立了20种氨基酸的高效液相色谱分离荧光检测及柱后质谱快速鉴定分析方法(HPLC-FLD-MS)，并实验验证了BCEC-Cl比以往报导的FMOC、CEOC、BCEOC氨基酸衍生试剂具有更高的紫外和荧光检测灵敏度。对波棱瓜子中的水解氨基酸和游离氨基酸进行了定量分析。 4、以1-(2-萘基)-3-甲基-5-吡唑啉酮(NMP)作为柱前衍生试剂，建立了9种单糖的高效液相色谱-DAD检测及在线质谱分析方法(HPLC-DAD-MS)，并验证了本法比传统单糖衍生试剂1-苯基-3-甲基-5-吡唑啉酮(PMP)的HPLC-UV法具有更高的紫外检测灵敏度。给出了NMP衍生物在ESI/MS条件下的分子离子裂解规律，标志性MS/MS碎片离子m/z 473.0是还原性单糖NMP衍生物裂解后的特征碎片离子。运用建立的新方法对波棱瓜子中的单糖进行了定性和定量研究。 5、建立了高效液相色谱-DAD检测-大气压化学电离质谱法(HPLC-DAD-APCI/MS)分离测定芦丁、槲皮素、山奈酚、异鼠李素四种黄酮的方法，方法的灵敏度高、线性范围宽、定性和定量准确，结合超声提取法和微波回流提取法，测定了波棱瓜子中水解和不水解的四种黄酮的含量。 6、对波棱瓜子油保护四氯化碳(CCl4)和D-半乳糖胺(D-GalN)化学性肝损伤进行了实验，结果表明，波棱瓜子油能显著抑制肝损伤所致的大鼠血清ALT、AST和MDA含量的升高，并能显著提高肝损伤大鼠血清的SOD活力，具有显著的保护化学性肝损伤作用。肝损伤大鼠肝组织病理学检验也同样表明波棱瓜子油具有保肝作用。这为波棱瓜子相关保肝药物的开发提供了科学依据。
其他摘要	Herpetospermum seed is the dried ripe seed of Herpetospermum pedunculosum (Ser.) Baill (H. Caudigerum Wall), which is produced in India, Nepal and China (Tibet, Qinghai province, Sichuan province, Yunnan province). As a commonly used Tibetan medicine, it is considered to possessing the functions of clearing the liver-fire and the gallbladder-heat, detoxifying and aiding the digestion. Therefore, it is frequently employed to curing liver heat syndrome, hepatitis A, hepatitis B and indigestion. Herpetospermum seed is a preferred medicine for liver disease in Tibetan traditional medicine, and it is frequently used in most Tibetan medicinal prescriptions for liver diseases. The seed oil of Herpetospermum pedunculosum (Ser.) Baill contains abundant unsaturated fatty acids, such as oleic acid, linoleic acid and linolenic acid. It is reported that unsaturated fatty acids can provide healthful physiological functions to human beings, such as protection effect for liver injury induced by chemical reagent, anti-oxidation, preventing tumour, improving immunity function, preventing heart disease and high blood pressure. Therefore, research in the seed of Herpetospermum pedunculosum (Ser.) Baill is of crucial importance to sufficiently utilize it for the exploitation of natural drugs. To develop new drugs with liver-protective effects, research in the composition of Herpetospermum seed using modern analytical technologies is in good agreement with the development law of Tibetan medicine, at the same time, it is also in favor of the popularity and application of it. In this research, the chemical components and the protective effect on chemical liver injury of Herpetospermum seed were investigated as the key research contents. The methods for the sensitive determination of fatty acids, amino acids and monosaccharides by pre-column derivatization with fluorescence detection and HPLC-MS were developed. In addition, a sensitive method for the determination of flavonoid compounds using HPLC-DAD-APCI/MS was also investigated. In view of the extracted Herpetospermum seed oil, the protective effect for liver injury induced by chemical reagent was evaluated by means of animal experiment. Some significant results and conclusions were obtained as follows. 1. Supercritical CO2 extraction technology was used to extract seed oil of Herpetospermum pedunculosum (Ser.) Baill, and the corresponding experimental parameters, such as extraction temperature, pressure, CO2 flux, extraction time and particle size of pulverized Herpetospermum seed were investigated. The optimum extraction conditions with the highest extraction yield were obtained as follows: extraction temperature 50℃, pressure 35 MPa, CO2 flux 35 L/h，80 mesh for particle size of pulverized Herpetospermum seed, extraction time 3.5 h. 2. A facile and sensitive method for the simultaneous determination of 30 saturated and 9 unsaturated fatty acids was developed by HPLC with fluorescence detection and on-line MS identification following pre-column derivatization with 1-[2- (p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP). The contents of fatty acids in the seed oil of Herpetospermum pedunculosum (Ser.) Baill from supercritical CO2 extraction, microwave-assisted reflux extraction and ultrasound-assisted extraction were compared. In addition, the acidic catalysis and alkaline catalysis of the methyl esterifying reaction were compared for the GC-MS analyzing fatty acids in Herpetospermum seed oil, and the characteristics of HPLC and GC-MS methods were discussed for the analysis of fatty acids. 3. A rapid and sensitive method for the determination of 20 amino acids was developed by HPLC with fluorescence detection and on-line MS identification following pre-column derivatization with 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl). The experimental results indicated that BCEC-Cl was more sensitive than the reported FMOC, CEOC and BCEOC for the determination of amino acids with fluorescence and UV. Using the established method, free and hydrolyzed amino acids in Herpetospermum seed were determined in this study. 4. A new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupling with HPLC-DAD detection and on-line electrospray ionization mass spectrometry (ESI-MS) for the detection of 9 monosaccharides was reported. The established method for the assay of monosaccharides using NMP as labeling reagent was more sensitive than the traditional HPLC-UV method using 1-phenyl-3-methyl-5-pyrazolone (PMP) as pre-column derivatization reagent. Each molecular ion of bis-NMP-monosaccharide derivatives presented abundant fragment ions. Among these ions the most intense and characteristic one at m/z 473.0 could be used for the accurately qualitative identification of monosaccharides. Using the established method, the monosaccharides in Herpetospermum seed were analyzed. 5. An HPLC-DAD-APCI/MS method was developed for the separation and determination of 4 flavonoid compounds (rutin, quercetin, kaempferol, isohamnetin). The method exhibited high sensitive, wide linearity range and good accuracy for the qualitation and quantitation analysis of flavonoids. The contents of hydrolyzed and non-hydrolyzed flavonoid compounds in Herpetospermum seed by ultrasound-assisted extraction and microwave-assisted extraction were determined. 6. The protective effect of Herpetospermum seed oil on acute CCl4- and D-GalN-induced liver injury was investigated using rats, and the results indicated that the Herpetospermum seed oil could remarkably inhibited the increase of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) in rat serum, and inhibited the decrease of the superoxide dismutase (SOD) activity induced by CCl4- and D-GalN. Furthermore, the pathological examination of the rat liver tissues also proved that the Herpetospermum seed oil had the protective effect on the rat chemical-induced liver injury. In conclusion, this research is to provid scientific foundation for the development of new drug with liver-protective effect from Herpetospermum seed oil.
页数	135
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3212
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	赵先恩. 藏药材波棱瓜子化学成分与保肝作用研究[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2009.

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