黄参籽精油、多酚的组成及其功能性研究

【作者】 高春燕；

【导师】 田呈瑞；

【作者基本信息】 陕西师范大学 ， 植物学， 2012， 博士

【摘要】 黄参(Sphallerocarpus gracilis),系伞形科,芹亚科,针果芹族,迷果芹属的单种植物迷果芹,多年生草本植物。其全草入药,是一种传统的藏药,有祛风除湿功效,主治风湿性关节炎。本文以甘肃山丹黄参籽为原料,研究了黄参籽精油和多酚的化学组成及功能性,对于天然抑菌剂和抗氧化剂的开发,黄参资源的利用与开发以及促进当地经济的发展都具有非常重要的现实意义。主要研究结果如下:1.不同提取方法所得黄参籽精油在化学组成、得率、颜色状态及气味方面差异较大。水蒸气蒸馏提取精油主要成分为对伞花烃、γ-松油烯和α-细辛醚,精油得率较低,呈无色透明液体,具强烈刺激性气味;超临界CO2流体萃取黄参籽精油主要成分为对伞花烃、γ-松油烯和α-细辛醚,精油得率较高,呈淡黄色透明液体,具较柔和刺激性气味;固相微萃取黄参籽精油主要成分为β-蒎烯、对伞花烃和γ-松油烯。另外,不同提取方法对黄参籽超微结构的影响不同,水蒸气蒸馏和超临界CO2流体萃取对黄参籽超微结构影响显著,组织结构均受到一定程度的破坏,而固相微萃取对黄参籽超微结构无明显的影响。2.不同提取方法所得黄参籽精油对微生物的抑制作用存在差异。对所测试的13种细菌,水蒸气蒸馏提取的精油对李斯特菌未表现出抑制作用,而超临界CO2流体萃取的精油对李斯特菌和藤黄微球菌未表现出抑制作用;对霉菌(黑曲霉)而言,水蒸气蒸馏提取和超临界CO2流体萃取的黄参籽精油都未表现出抑制作用。大体上讲,水蒸气蒸馏法提取的黄参籽精油其抑菌活性强于超临界CO2流体萃取的黄参籽精油。供试菌种中,黄参籽精油对克雷伯肺炎杆菌的抑制作用最强。3.黄参籽精油成分可以影响微生物的生长曲线,对微生物的萌发有明显的阻滞作用;对微生物细胞的表面结构均有不同程度的破坏,形态学上发生了较大改变,主要表现为微生物细胞壁变形,细胞内容物泄露及细胞表面粗糙、有附着物,菌体细胞发生断裂及微生物细胞溶解;此外,黄参籽精油对微生物细胞的内部结构也有一定程度的破坏作用,经黄参籽精油处理后,菌体失去其完整结构,细胞壁被破坏,胞间质层模糊不清,不能清晰的分辨细胞壁和细胞膜,胞质不均匀,且有些细胞内,细胞质甚至解体出现空泡结构。4.黄参根、茎、籽中总酚含量不同,其中籽含量最高,其次是茎,根含量最低;游离酚、可溶性糖甙结合酚、可溶性酯结合酚及不溶性细胞壁结合酚在黄参根、茎、籽中含量存在差异;另外,不同采收期黄参根的酚含量也不同,春季采收的酚含量较高。5.对黄参籽中含量较高的9种酚类物质进行鉴定,分别为绿原酸、咖啡酸、芍药花素二葡萄糖甙、木犀草素葡萄糖甙、二咖啡酰奎尼酸葡萄糖甙、儿茶酸葡萄糖甙,且在黄参籽游离酚、可溶性糖甙结合酚、可溶性酯结合酚及不溶性细胞壁结合酚提取物中其含量是有差异的。6.黄参籽游离酚、可溶性糖甙结合酚、可溶性酯结合酚和不溶性细胞壁结合酚提取物具有明显的铁还原力、抑制定位和非定位羟基自由基介导的2-脱氧-D-核糖裂解的作用以及清除DPPH和ABTS自由基的能力,且不同部分的多酚提取物在各个体系中的抗氧化能力呈现多样性。7.黄参籽多酚提取物酚含量与DPPH自由基清除活性、FRAP和TEAC呈显著的正相关,但是与抑制非定位和定位羟基自由基介导的2-脱氧-D-核糖裂解作用无显著正相关;不同抗氧化体系之间,DPPH自由基清除活性、FRAP与TEAC呈显著正相关,对非定位和定位羟基自由基介导的2-脱氧-D-核糖裂解的抑制作用显著相关;相反,对羟基自由基介导的2-脱氧-D-核糖裂解的抑制作用与DPPH自由基清除活性、FRAP和TEAC无明显的相关性。8.黄参籽游离酚、可溶性糖甙结合酚、可溶性酯结合酚和不溶性细胞壁结合酚提取物对·OH和ROO·介导的pBR322质粒DNA的损伤都具有一定的保护作用,且对·OH介导的DNA损伤的保护作用强于ROO·。相关性分析表明,黄参籽多酚提取物对·OH和·OOR介导的DNA损伤保护作用不仅与酚含量有关,而且与酚提取物的组成成分有关。9.黄参籽游离酚、可溶性糖甙结合酚、可溶性酯结合酚和不溶性细胞壁结合酚提取物对人结肠癌细胞Lovo的增殖具有明显的抑制作用,且随着酚浓度的增加,抑制率上升。形态学的观察结果表明,经黄参籽多酚提取物处理后细胞形态发生明显改变,细胞固缩、干瘪,体积变小,甚至干枯脱落,初步提示黄参籽多酚可以诱导人结肠癌细胞Lovo的凋亡。10.黄参籽游离酚提取物对CCl4所致小鼠肝损伤具有一定的保护作用,能显著抑制CCl4所致肝损伤小鼠肝脏指数、脾脏指数、血清AST、ALT和AKP活性以及肝脏TG、TC和MDA水平的升高,能提高肝脏抗氧化酶SOD、CAT和GSH-PX的活性,其中以中剂量效果最佳。黄参籽游离酚提取物中剂量组小鼠肝细胞病变较轻,细胞只有点状坏死,部分水肿,无大面积的凝固性坏死,进一步说明了对CC14所致小鼠肝损伤的保护作用。更多还原

【Abstract】 Sphallerocarpus gracilis(S.gracilis)is a perennial member of the Apiaceae family.According to ancient records,the entire S.gracilis plant was widely used to treat rheumatic arthritis,tetanus and ulcers in traditional Chinese medicine.The chemical composition and functionality of essential oil and phenolic compounds in S.gracilis seed were studied in this paper,which has a very important practical significance for the development of natural antibacterial agents and antioxidants,utilization and develpoment of S.gracilis and promotion of the local economics.The main research results of this paper are as follows:1.The chemical composition,yield,color,state and flavor of the essential oil from S.gracilis seed varied greatly with the extraction methods.The essential oil obtained by steam distillation was colorless,pungent with a lower yield,and its main constituents were p-cymene,y-terpinene and a-asarone.The essential oil extracted by supercritical carbon dioxide was light yellow,soften with a higher yield and its major components were p-cymene,γ-terpinene and a-asarone.The main constituents of the essential oil extracted by solid-phase microextraction were β-Pinene,p-cymene and γ-terpinene.In addition,the ultrastructure of the S.gracilis seed varied greatly with the different extraction methods.The ultrastructure of the S.gracilis seed treated with steam distillation and supercritical carbon dioxide extraction was greatly damaged.Whereas,no significant destroy was observed of the S.gracilis seed treated with solid-phase microextraction.2.The antibacterial activity of the essential oil from S.gracilis seed varied greatly with the extraction methods.Among the 13 tested microorganisms,no inhibitory effect was found against L.monocytogenes of the essential oil obtained by steam distillation,whereas no inhibitory effects were observed against L.monocytogenes and M.luteus of the essential oil obtained by supercritical carbon dioxide.As far as the A.niger was concerned,the essential oil extracted by steam distillation and supercritical carbon dioxide showed no inhitory effect.As a whole,the antibacterial activity of the essential oil obtained by steam distillation was stronger than the the essential oil extracted by supercritical carbon dioxide.Among all the tested microorganisms,the essential oil from S.gracilis seed showed the strongest inhibitory effect against K.pneumoniae.3.The components of the essential oil from S.gracilis seed effected the growth cruve of microorganisms and inhibited the germination of microorganisms.Physical damage and considerable morphological alteration were observed to the tested microorganisms treated with the essential oil.The cells present deformity in the cell walls,leakage of intracelluar constituents,surface roughness,fragmentation and lysis.Additionally,the cellular ultrastructure were also damaged by the essential oil from S.gracilis seed.The cell losted its whole structure and cell wall was destoried.Additionally,it was difficult to distinguish cell membrane and cell wall and the cytoplasm was not homogeneous.In some cells,the cytoplasm showed a serious collapse and vacuoles structure was observed.4.The total phenolic content in S.gracilis root,stem and seed was different.Higher levels of phenolic content was detected in seed,followed by stem and root;The content of free,soluble glycoside,conjugated and insoluble cell-wall-bound phenolics was different in S.gracilis root,stem and seed.In addition,the phenolic content of the S.gracilis root varied greatly with the harvest time and higher amounts of phenolic content was found in root harvested in spring.5.Chlorogenic acid,caffeic acid,peonidin-diglucoside,luteolin-glucoside,di-caffeoylquinic acid glucoside and catechin glucoside were identified in S.gracilis seed and their contents in free,soluble glycoside,conjugated and insoluble cell-wall-bound phenolic extracts were different.6.Significant ferric reducing antioxidant power,inhibitory effects on site and non-site hydroxyl radical mediated 2-deoxy-D-ribose degradation and scavenging capacity of DPPH and ABTS radicals were found of the free,soluble glycoside,conjugated and insoluble cell-wall-bound phenolic extracts from S.gracilis seed.Furthermore,the antioxidant activities of different phenolic extracts in different antioxidant systerms were various.7.The data demonstrated that significant correlation was found between phenolic content and DPPH radicals scavenging capacity,FRAP and TEAC.Conversely,no significant correlation was observed between phenolic content and inhibitory effects on site and non-site hydroxyl radical mediated 2-deoxy-D-ribose degradation.Among the methods used for quantifying antioxidant activity,significant correlation was found between DPPH radicals scavenging capacity,FRAP and TEAC,and inhibitory effects on site and non-site hydroxyl radical mediated 2-deoxy-D-ribose degradation.In contrast,there was no significant correlation between inhibitory effects on hydroxyl radical mediated 2-deoxy-D-ribose degradation and DPPH radicals scavenging capacity,FRAP and TEAC.8.Significant-OH and ROO· mediated DNA damage protective effects were observed of free,soluble glycoside,conjugated and insoluble cell-wall-bound phenolic extracts from S.gracilis seed.Moreover,-OH mediated DNA damage protective effects of the phenolic extracts higher than the ROO-mediated DNA damage.Results of the correlation analysis indicated that ·OH and ROO· mediated DNA damage protective effects of the phenolic extracts from S.gracilis seed not only depended on the phenolic content but also the type of phenolic constituent present in each fraction.9.Significant inhibitory effects were found of free,soluble glycoside,conjugated and insoluble cell-wall-bound phenolic extracts from S.gracilis seed on the Human Colon Carcinoma Lovo Cells.Furthermore,the inhibition rate gradually enhanced followed by the phenolic concentrations increasing.Based on the morphological observation,the morphology of the cells treated with the phenolic extracts changed greatly.Cell shrinkage and nuclear condensation were observed,which suggested that phenolic extracts from S.gracilis seed can induce the Human Colon Carcinoma Lovo Cells apoptosis.10.Results indicated that the free phenolic extract from S.gracilis seed showed preventive effect on liver injury induced by CCl4.The phenolic extract from S.gracilis seed remarkably inhibited the increase of liver index,spleen index,the activities of serum ALT,AST,AKP and liver TG,TC,MDA content,and elevated activities of SOD、CAT and GSH-PX induced by CCl4.Moreover,the middle dose group is the most advantage.The mice hepatic cells treated with middle dose were slightly damaged with a little intense coagulation necrosis,which further showed the hepatoprotective effects of the phenolic extract against carbon tetrachloride induced liver injury in rats.更多还原