【作者】 丁友芳；

【导师】 刘玉军；

【作者基本信息】 北京林业大学 ， 植物学， 2012， 博士

【摘要】 紫苏[Perilla frutescens (L.) Britt.]为唇形科(Labiatae)紫苏属(Perilla)一年生草本植物,是我国传统的药食两用植物,资源丰富。紫苏籽,又称苏子,是紫苏的干燥成熟果实,为中国药典所收载,具有降气消痰、平喘、润肠等功效。本文以紫苏种子为研究对象,对其质量分级标准、品质特性,含油率和脂肪酸组成以及提取物的化学组成和生物活性进行了系统的研究,主要结果如下：1紫苏种子质量分级标准研究通过净度分析、真实性鉴定、健康检验、重量测定、水分测定、生活力测定、发芽率测定等研究,制定了紫苏种子检验规程；通过对国内50份不同产地的紫苏种子的净度、千粒重、含水量、生活力以及发芽率检测和K-聚类分析,将紫苏种子划分为3个等级：1级种子的发芽率≥84.94%,净度≥92.44%,千粒重≤1.87g,含水量≤8%；2级种子发芽率56.44～84.94%,净度92.35～92.44%,千粒重1.87～2.06g,含水量≤8%；3级种子发芽率29.50～56.44%,净度90.95～92.35%,千粒重2.06～3.05g,含水水量≤8%。2紫苏种子含油率及脂肪酸组成研究对不同产地5个紫苏变种种子的品质性状、含油率以及脂肪酸组成等开展的比较研究表明,5个变种种子的形状、颜色、纹理结构基本无差异,但粒径大小、含水量、千粒重、含油率有极显著差异。白苏变种种子平均粒径较大,野生紫苏变种和回回苏变种籽粒较小；千粒重、含水率从大到小依次为白苏>耳齿紫苏>紫苏>野生紫苏>回回苏；种子的含油率由高到低依次为耳齿紫苏、紫苏、野生紫苏、回回苏、白苏。5个紫苏变种种子油中主要脂肪酸均为α-亚麻酸、油酸、亚油酸、棕榈酸等9种组分,但不同产地紫苏种子中各种脂肪酸的含量及比例不同,其中α-亚麻酸含量由高到低依次为耳齿紫苏、紫苏、回回苏、野生紫苏、白苏；以上结果表明,不同产地变种紫苏种子品质性状、含油率及脂肪酸组成存在较大差异,其中云南文山耳齿紫苏种子品质最优,推测紫苏种子品质性状,含油率以及脂肪酸组成等受海拔、纬度和年平均温度等环境因素的影响较大。3紫苏饼乙醇和水提取物抗氧化活性比较研究对紫苏种子榨油后的残渣(紫苏饼)依次进行了石油醚脱脂、乙醇提取和水提取。其中乙醇和水提取物得率分别为6.15%和8.20%；醇提物中总酚和总黄酮含量分别为83.01mg/g和30.41mg/g,主要单体成分为咖啡酸、迷迭香酸和木犀草素等酚酸成分；水提物中多糖成分含量为38.58%。紫苏饼醇提物和水提物的体外抗氧化实验表明,其对DPPH自由基、超氧阴离子(O2)自由基、羟基自由基(·OH)均有清除作用,且具良好的量效关系；乙醇提取物对DPPH自由基、·OH和O2的清除ECso值分别为113.264、266.693和1411.183μg/mL,水提取物的ECso值分别为349.694、1076.04和1154.615μg/mL,表明紫苏饼醇提物比水提物具有更强的抗氧化活性。进一步研究表明,紫苏饼乙醇提取物添加于紫苏油中能有效降低紫苏油的脂质氧化水平,提高氧化稳定性,在添加量为0.06%时,其抗氧化活性与BHT(0.02%)相当。4紫苏饼乙醇提取物抗炎作用及其机制研究采用二甲苯致小鼠耳肿胀和角叉菜胶致小鼠足肿胀炎症模型考察了紫苏饼乙醇提取物的抗炎活性,发现其醇提物对二甲苯致小鼠耳肿胀和角叉菜胶所致小鼠足肿胀均有明显的抑制作用,且呈显著的量效关系,表明醇提物具有明确的抗炎作用；同时发现,醇提物可显著抑制角叉菜胶诱发的小鼠血清中促炎细胞因子IL-6和TNF-α含量的增加,降低炎症渗出液中PGE2和MDA的含量,提高小鼠血清中SOD的活力,推测紫苏饼醇提物的抗炎作用可能是通过抑制COX-2活性及体内氧化损伤而实现的。5紫苏饼乙醇提取物急性毒性与遗传毒性研究小鼠急性毒性实验中,24小时内给予小鼠20g/Kg.BW剂量的紫苏饼乙醇提取物,7d内未发现实验小鼠出现明显中毒症状和死亡病例,表明醇提物的LD5o远大于20g/Kg.BW,属于实际无毒级。Ames实验中,不同剂量受试物(醇提物)在加S9和不加S9条件下的菌落回复突变数均未超过空白对照组和溶剂对照组的两倍,且各剂量组间无明显的剂量反应关系；小鼠骨髓嗜多染红细胞微核实验中,醇提物各剂量组小鼠骨髓嗜多染红细胞微核率明显低于阳性对照组,且与阴性对照组无显著性差异；精子畸形实验中,醇提物各剂量组精子畸形率明显低于阳性对照组(p<0.01),且与阴性对照组无显著性差异。以上结果表明,醇提物无明显的急性毒性和致突变作用,是相对安全的。更多还原

【Abstract】 Perilla frutescens (L.) Britt. is an annual herbaceous plant in the family Labiatae. It is also a traditional food and medicinal plant widely distributed in China. According to Chinese Pharmacopoeias (2005edition), Perilla seeds, as the ripe fruits of the plant, have many potential therapeutic properties, such as lowering adverse-rising energy, dispersing phlegm, relieving asthma, moistening intestine and so on. In this article, we systematically investigated quality classification standards, agronomic characters, oil contents, and fatty acid compositions of the Perilla seeds, and the chemical constituents and bioactivities of extracts from Perilla seed residues after extracting of the oils. Main findings are as follows:1Quality classification standards of the Perilla seedsInspection rules for Perilla seeds were established based on investigation results of purity analysis, authenticity identification, health test, weight determination, moisture determination, viability test and germination rate test. According to the results of purity,1000-seed weight, moisture content, viability, germination rate and K-clustering analysis of Perilla seeds of fifty origins from all over the China, Perilla seeds were divided into three grades:for the first grade, germination rate>84.94%, purity≥92.44%,1000-seed weight≤1.87g, moisture content<8%; for the second grade, germination rate between56.44%and84.94%, purity between92.35%and92.44%,1000-seed weight between1.87g and2.06g, and the moisture content<8%; for the third grade, germination rate between29.50%and56.44%, purity between90.95%and92.35%,1000-seed weight between2.06g and3.05g, and the moisture content<8%.2Oil contents and fatty acid compositions of the Perilla seedsBy comparing the agronomic characters, oil contents and fatty acid compositions of seeds of five Perilla variants from five different origins in China, it is shown that there was no difference in shape, color and texture. However, significant differences were indeed found in size, moisture content,1000-seed weight and oil content. In detail, average grain diameter of var. frutescens seeds was larger than others, while var. acuta and var. crispa had the smallest average grain diameter. Var. frutescens had the highest1000-seed weight and moisture content, followed by var. auriculato-dentata, var. arguta, var. acuta and var. crispa. For oil content, var. auriculato-dentata had the highest value, followed by var. arguta, var. acuta, var. crispa and var. frutescens. The USFA content in seeds of all the five variants was above90%, consisted of nine fatty acids, including a-linolenic acid, oleic acid, linoleic acid, palmitic acid and so on. However, the content and ratio of each fatty acid in seeds from different regions were significantly different. Var. auriculato-dentata possessed the highest content of a-linolenic acid, followed by var. arguta, var. crispa, var. acuta and var. frutescens. The results indicated that the quality characteristic, oil content and fatty acid compositions of Perilla seeds were substantially affected by environmental factors such as elevation, latitude, and average annual temperature. For Perilla seeds from different regions, significant differences in quality characteristics, oil contents and fatty acid compositions were found. The seed of var. auriculato-dentata from Yunnan Wenshan has the best quality among all five seeds tested.3Comparison of anti-oxidant activities of ethanol and water extracts from Perilla seed residuesPerilla seed residues after oil extraction were defatted by petroleum ether, extracted by ethanol and water successively. The yields of ethanol and water extracts were6.15%and8.20%, respectively. In ethanol extract, total polyphenols and flavonoids were83.01mg/g and30.41mg/g, respectively. Caffeic acid, rosmarinic acid and luteolin were the three major phenolic acids in the ethanol extracts, and the water extract contained38.58%of polysaccharide.Antioxidant experiments indicated that both ethanol and water extracts from the Perilla seed resiues possessed DPPH O2-and·OH free radicals scavenging activities with a dose-dependent manner. For the three kinds of free radicals, EC50were113.264,266.693and1411.183ug/mL in ethanol extract, and349.694,1076.04and1154.615μg/mL in water extract, respectively. It is clear that the antioxidant activities of ethanol extract was stronger than those of water extract. Further studies showed that addition of the ethanol extract into the Perilla seed oil could effectively reduce the lipid oxidation level and improve its oxidative stability. As antioxidant additives, the effect of0.06%ethanol extract is equivalent to0.02%of BHT in oils.4Anti-inflammatory activity and mechanism of ethanol extract of Perilla seed residuesThe anti-inflammatory activity of ethanol extract was examined using inflammatory models including both the ear edema induced by xylene and the paw edema induced by carrageenan in mice. Results show that ethanol extracts obviously inhibited ear and paw edemas of mice and a significant dose-effect relationship was observed. Thus, it is clear that ethanol extracts possessed positive an anti-inflammatory activity. Ethanol extracts was also observed to significantly inhibit the increase of proinflammatory cytokines (IL-6and TNF-α) in mice serum induced by carrageenan, reduce the content of PGE2and MDA in inflammatory exudate fluid and enhance the activity of SOD in mice serum. It can be inferred that ethanol extracts play a role in anti-inflammatory action by suppressing COX-2activity and oxidative damage in vivo.5Acute and genetic toxicities of ethanol extract of Perilla seed residuesIn acute toxicity test, no obvious symptom of poisoning and death was observed within seven days after20g/Kg BW ethanol extract of Perilla seed residues was orally administrated in mice, indicating that LD50value of ethanol extracts was far greater than20g/Kg BW. In Ames test with or without S9fractions, the numbers of revertant colonies for different doses groups of ethanol extracts were no more than twice of those for both the blank and solvent control groups, and no obvious dose-response relationship was observed. In polychromatic erythrocytes (PCE) of the mouse bone marrow test, the rate for PCE of the mice bone marrow in different doses of ethanol extracts groups was obviously lower than that of the positive control group, and there was no significant difference between different doses of ethanol extracts groups and the negative control group. In mice sperm malformation test, the rate of mice sperm malformation in different doses of ethanol extracts groups was significantly lower than that in positive control group (p<0.01), and there was also no significant difference between different doses of ethanol extracts groups and the negative control group. These results indicated that ethanol extracts has no acute toxicity and mutagenic effects, therefore, it is relatively a safe product.更多还原