【作者】 倪勤学；

【导师】 张英； 方伟；

【作者基本信息】 浙江大学 ， 食品科学， 2013， 博士

【摘要】 核辐射已广泛应用于军事、能源、医疗、农业等领域,在带给我们便利的同时也带来了安全隐患。安全高效的天然辐射防护剂的开发具有非常重要的现实和战略意义。竹叶富含次生代谢产物,具有优良的清除自由基、抗辐射、抗衰老、抗菌、抗病毒、提高免疫力等生物学功效。前期已有体外研究表明竹叶黄酮(毛金竹叶醇提物的正丁醇分极组分,代码为BLF-d24)试样浓度在20～160μg/mL时,对60Co-γ射线(O-20Gy)辐照剂量下的DNA损伤具有保护作用,但其在整体动物水平上的辐射损伤防护作用未见报道。迄今为止,对竹叶有效成分的研究和开发主要集中在以刚竹属毛金竹[Phyllostachys nigra (Lodd. ex Lindl.) Munro var. henonis (Mitford) Stapf ex Rendle]叶为代表的经济竹种上。刚竹属品种是我国最重要的经济竹种,占了资源量的70%以上,其秆型高大,叶片细小,主要以材用林、笋用林和笋材两用林为主,从竹叶的生物量大小和采集的便利程度看并无优势,亟须研发专用的叶用竹林。本课题正是基于这种产业背景,瞄准秆形矮小、叶子密集、生物量大、适于机械化采摘的地被竹(俗称竹草,Bamboo grass)种,选取鹅毛竹(Shibataea chinensis Nakai)、黄条金刚竹(Pleioblastus kongosanensis f. aureostriatus Muroi et Yuk. Tanaka)、阔叶箬竹[Indocalamus latifolius (Keng) McClure]、翠竹[Sasa pygmaea (Miq.) E. G.Camus]、铺地竹(Sasa argenteastriatus E.G.Camus)和隈笸[Sasa Veitchii (Carr.) Rehd.]六个品种,研究其竹叶中有效成分的含量及其抗氧化活性和对DNA损伤的防护能力；选取其中最具代表性和开发潜力的黄条金刚竹,分离制备其有效活性组分和特征性化学成分,进行结构解析；并对黄条金刚竹叶的活性组分进行辐射防护的动物试验研究,探索其防护作用的机制。主要结果归纳如下：(1)对六种地被竹叶的有效成分和生物学活性进行了比较。于2010年5月对位于临安市浙江农林大学翠竹园内的6种地被竹(鹅毛竹、黄条金刚竹、阔叶箬竹、翠竹、铺地竹、隈笹)叶进行同步采样,热风干燥后,用70%的乙醇热回流提取,制备竹叶提取物,用比色法测定其总黄酮、总酚和三萜含量,表明其均含有丰富的次生代谢产物,以叶干基计的有效成分含量变化在总酚4.15-9.12%、总黄酮1.62-4.00%、三萜1.54-2.28%之间。其中总酚含量最高的是鹅毛竹(9.12%),其次是黄条金刚竹(5.51%),二者的总黄酮含量分别为4.00%和3.16%,三萜含量分别为2.28%和1.89%；采用DPPH自由基清除能力和铁离子还原法(FRAP)两种抗氧化评价体系以及琼脂糖凝胶电泳法,对上述6种竹叶提取物进行抗氧化活性和DNA损伤防护能力评价,鹅毛竹和黄条金刚竹显著优于其他地被竹种(p<0.05)。综合考虑叶片生物量及片型大小、大面积栽培的推广潜力等因素,确定以黄条金刚竹作为进一步研究的对象。(2)对黄条金刚竹的生物量大小进行了研究。结果显示黄条金刚竹种群2010年8月的现存生物量为2428.18g/m2,其中地上部分(叶和秆)为1629.97g/m2,占总生物量的67.13%；地下部分(竹鞭)为798.21g/m2,占总生物量的32.87%。最有意义的是黄条金刚竹的叶生物量为746.53g/m2,为刚竹属竹种如毛竹叶生物量(350.5g/m2)的2.13倍,具有开发叶用竹林的潜力。(3)研究了季节变化和不同的前处理方法对黄条金刚竹叶有效成分含量及抗氧化活性的影响。结果显示,秋冬季节(9月-12月)采收的竹叶有效成分含量最高、抗氧化活性最强。2011年9月采收的黄条金刚竹叶样经100℃热风干燥,在料液比1：15、温度70℃、热回流提取1h的条件下,得到的70%醇提液(折算成竹叶干基计,下同)总酚含量为3.15%、总黄酮1.98%、三萜2.28%,清除DPPH自由基的IC50值为502.0μg/mL)。显著高于其他干燥方法(如阴干、晒干、微波处理干燥等)(p<0.05)。对黄条金刚竹叶的醇提工艺参数进行了优化,适宜的提取条件为：提取温度70℃,料液比1：25,乙醇浓度70%,提取时间80min；采用优化后的工艺参数组合得到的醇提液清除DPPH自由基的活性提高了19.46%(IC50值为404.32μg/mL)。此外,还探索了采用紫外线辐照处理鲜叶以增加次生代谢产物的含量,短波长(254nm)、低剂量(1kJ/m2)的紫外辐照处理能使竹叶总酚含量增加56.66%、总黄酮含量增加41.94%,清除DPPH自由基能力提高10.30%,表明紫外辐照处理是一种便捷、经济、有效、适合规模化生产的前处理方法。(4)从有效成分含量和生物学活性两方面筛选了黄条金刚竹叶的有效活性组分。70%乙醇提取物及其4个分极组分(石油醚相、乙酸乙酯相、正丁醇相及水分极相)均表现出对DPPH·、 ABTS·+、·OH、O·2-、H2O2显著的清除作用和较强的还原力,并对·OH和60Co-γ射线辐射造成DNA的损伤具有良好的防护能力。其中,正丁醇相的总黄酮含量(18.97%)和得率(占醇提物干基质量的7.49%)最高,乙酸乙酯相的总酚含量(32.42%)和三萜含量(7.37%)最高;这两相均表现出强于其他分极组分的抗氧化活性和DNA损伤防护能力(p<0.05)。鉴于此,在实际应用中,将这两个中等极性相(正丁相和乙酸乙酯相)合并,即黄条金刚竹叶的70%乙醇提取物经石油醚脱脂后,直接用正丁醇进行液-液萃取,得到黄条金刚竹叶的活性组分(PLE-EP).(5)对黄条金刚竹叶的特征性成分进行分离、制备、结构解析和活性比较。采用正相和反相硅胶柱、RP-C18. Sephadex LH-20柱层析和薄层色谱,从上述活性组分(PLE-EP)中分离制备得到了5个化合物单体,通过紫外光谱、质谱、核磁共振技术,鉴定其化学结构分别为对香豆酸、阿魏酸、kurilensin A[木犀草素6-C-a-阿拉伯呋喃糖基(1→2)-a-L-吡喃鼠李糖苷]、siparunoside[山奈酚-3,7-双甲氧基-4,-O-β-[α-鼠李糖(1→6)-葡萄糖苷]和苜蓿素,RP-HPLC去测得PLE-EP中上述化合物的含量分别为11.30、2.05、12.64、3.18和8.79mg/g。5个化合物单体均呈现出对DPPH·、ABTS+良好的清除活性和较强的还原力,其中,kurilensin A的抗氧化活性可与Vc相媲美,显著高于其他4个化合物(p<0.05)；同时,kurilensin A具有显著的对γ-射线造成DNA辐射损伤的防护能力,当试样浓度为100μg/mL时,对质粒DNA辐照损伤的抑制率达到75.0%。表明kurilensin A是黄条金刚竹叶抗氧化和抗辐射的关键成分,可作为黄条金刚竹叶提取物的特征性成分。(6)黄条金刚竹叶活性组分防护辐射损伤的动物试验研究。采用60Co-γ射线辐射挣伤小鼠模型,比较了PLE-EP与目前市售的竹叶黄酮制剂(BLF-d24)对小鼠辐射损伤的防护能力。结果显示,两者在200mg/kg·d的给药剂量下均可提高7Gy辐照剂量小鼠的30d存活率,保护指数均为1.44。5Gy辐照剂量可导致小鼠外周血象不同程度的变化,灌胃不同剂量的PLE-EP (100,200.400mg/kg-d)和BLF-d24(200mg/kg·d)可浓度依赖地减缓辐照小鼠血液中白细胞、红细胞、血小板和血红蛋白的下降幅度,特别是给药3周后,表现出显著的升白作用(p<0.05)；与辐照对照组比较,高剂量(400mg/kg-d) PLE-EP能使辐照小鼠的骨髓有核细胞数恢复至正常水平,骨髓嗜多染红细胞微核率显著下降(p<0.05)。同时,PLE-EP和BLF-d24均可显著提高辐照小鼠血清、肝脏、脾脏和胸腺组织中SOD、GSH-Px、CAT等抗氧化酶的活性,降低MDA、LPO、LF等氧化产物含量,具有增强小鼠抗氧化体系的功能,并对辐照引起的肝脏、胸腺和脾脏等脏器的损伤有不同程度的防护作用。综合评价,PLE-EP和BLF-d24的辐射防护效果相当。综上所述,黄条金刚竹叶提取物具有良好的抗氧化活性和DNA损伤防护能力,而且在动物体的辐射损伤防护上表现出良好的效果,其主要有效成分主要为酚酸和黄酮类化合物,与刚竹属的毛金竹叶提取物基本一致；香豆酸、阿魏酸、kurilensin A、siparunoside和苜蓿素是首次在黄条金刚竹叶中检出；kurilensin A仅在Sasa属的竹草中有过报道,迄今未见在刚竹属竹子中检出,而siparunoside则在竹亚科植物中未见报道。此外,黄条金刚竹生长速度快、叶生物量大,具有作为开发叶用竹林培育和开发的优势。本文研究结果为竹叶化学素和竹类资源的现代生物利用拓展了新的空间。更多还原

【Abstract】 Nuclear radiation is widely used in the filed of military, energy, health, agriculture etc, however, it is also of tromendous concern in terms of public security. Developing safe and effective radiation protectant is of great importance. The bamboo leaves, with diversed secondary metabolites, have been shown to have significant biological effects such as free radical scavenging activity, anti-radiation, anti-aging, anti-bacterial, anti-viral, enhancing immunity and others. Recent studies showed that at concentration of20～160μg/mL, bamboo leaf flavone [butanol fraction from70%ethanol extract of Phyllostachys nigra var. hnonis (Bean) Stepf ex Rendle] could prevent DNA from60Co-y irradiation damage. However, this radioprotection effects have not been confirmed by in vivo studies. Till now, the utilization of bamboo leaves focus mainly on family Phyllostachys, which has tall stem and small leaves and is difficult to harvest. The present study is designed to analyze the radioprotection effects of several bamboos grasses, namely Shibataea chinensis Nakai, Pleioblastus kongosanensis f. aureostriatus Muroi et Yuk Tanaka, Indocalamus latifolius (Keng) McClure, Sasa pygmaea (Miq.) E. G. Camus, Sasa argenteastriatus E.G. Camus and Sasa veitchii (Carr.) Rehd. These bamboo grasses have abundant active compounds and high antioxidant activity, meanwhile, they have rapid growth rate, lush foliage and are easy to harvest. Therefore, they have the potential to be developed as a kind of leafy bamboo grove. Firstly, the content of active components, antioxidant activities and DNA damage prevention abiligy of the above six bamboo grasses leaf were investigated. Then, the most reprensative and potential P. kongosanensis f. aureostriatus was selected. Its effective parts of the selected bamboo grass were screened by detecting the active components content and relalated chemical compounds. And finally, the study of radioprotection ability of the effective parts was carried out and its protective functions were also determined. The main conclusions are as follows:(1) The content of active components, antioxidant activities and DNA damage prevention ability of the above six bamboo grasses leaves were compared. S. chinensis and P. kongosanensis f. aureostriatus contains higher total phenolic (9.12% and5.51%, respectively), total flavonoids (4.00%and3.16%, respectively) and titerpenoids (2.28%and1.89%, respectively) content than the others. The antioxidant activity and DNA damage prevention ability of those two species were also stronger than others (p<0.05). Taking into account factors such as leaf size, collection and cultivation, P. kongosanensis f. aureostriatus with the advantage of larger leaf, easier picking and wider range of cultivation, was selected as the further research object.(2) The biomass of P. kongosanensis f. aureostriaus was observed. Results show that the leaf biomass of P. kongosanensis f. aureostriaus was746.53g/m2, which is of2.13times of that of Phyllostachys pubescens Mazel ex H. de Lehaie (350.5g/m2). It means P. kongosanensis f. aureostriaus have the advantage to be developed as a kind of leafy bamboo grove.(3) Changes of the antioxidant activity and active compounds content from P. kongosanensis f. aureostriatus affected by different harvest season and pre-treatment were investigated. Results showed that bamboo grasses leaf have the highest level of total active components and antioxidant activity in autumn-winter. Different drying methods also affected the active compounds. The bamboo leaf which is collected from September and treated by hot air drying contained significant higher total phenolic (3.15%), total flavonoids (1.98%), titerpenoids (2.28%) and stronger DPPH radical scavenging ability (IC50502.00μg/mL, DW) than other drying methods, i.e. shade drying, sunny drying and microwave drying (p<0.05). The DPPH radical scavenging IC50reached404.32μg/mL by using an extraction temperature70℃, material/liquid ratio1:25, extraction duration80min and the ethanol concentration70%. Additionally, UV treatment of low dosage (1kJ/m2) which can effectively enhance the content of total phenolic56.66%, total flavonoids41.94%, DPPH radical scavenging ability10.30%, is a convenient, economical and effective pre-treatment method for bamboo leaf.(4) The effective part of P. kongosanensis f. aureostriatus leaf extract was selected by active components content and biological activity determination. The70%ethanol extract of P. kongosanensis f. aureostriatus (PLE) and its four different polarity fractions i.e. petroleum ether fraction (PLE-PF), ethyl acetate fraction (PLE-EF),n-butanol fraction (PLE-BF) and water fraction (PLE-WF) showed concentration dependent scavenging ability on DPP·、ABTS·+、·OH、O·2-、H2O2and DNA damage prevention ability. PLE-BF had the highest yield rate (7.49%, DW) and total flavonoids content (18.97%) while PLE-EF possessed the highest content of total phenolic (32.42%) and titerpenoids (7.37%). Both two fractions showed the highest antioxidant activity and DNA damage prevention ability than others (p<0.05). Thus, in practice, PLE-BF and PLE-EF were combined and supposed to be the effective part of P. kongosanensis f. aureostriatus leaf extract (PLE-EP).(5) The characteristic compounds of P. kongosanensis f. aureostriatus leaf were isolated, purified and structural analyzed. By using normal phase and reversed-phase silica gel column, RP-C18, Sephadex LH-20and thin-layer chromatography, five compounds were isolated from PLE-EP. They were identified as p-coumaric acid, ferulic acid, kurilensin A, siparunoside and tricin. The contents of them were measured by RP-HPLC, with a value of11.30,2.05,12.64,3.18and8.79mg/g in PLE-EP. Free radical scavenging effect (DPPH·, ABTS·+), reducing power and DNA damage prevention ability of each compounds were also investigated. Kurilensin A showed significant stronger antioxidant activity and DNA damage prevention ability than others (p<0.05), was referred to be the main active compound in P. kongosanensis f. aureostriatus leaf.(6) The radioprotection ability of P. kongosanensis f. aureostriatus leaf were determined using animal test. In the60Co.y radiation injured mice, both PLE-EP and bamboo leaf flavonoids (BLF-d24) was able to significantly improve the survival of the treated mice in the30d test at the dose of200mg/kg-d. Also, they decreases the white blood cell, red blood cell, platelet counts and hemoglobin level in the peripheral blood of the radiation injured mice. After3weeks administration, the white blood cell counts of radiation injured mice was significantly improved (p<0.05). Additionally, PLE-EP (400mg/kg-d) was able to rescue nucleated cells in treated mice bone marrow to normal level and significantly reduce its micronucleated polychromatic erythrocytes. Futhermore. both PLE-EP and BLF-d24was shown to elevate the endogenous antioxidant enzymes (SOD、GSH-Px、CAT) activities and reduce the MDA、LPO、LF content, and protect the liver, thymus and spleen of mice from irradiation injury to different degrees.In conclusion, P. kongosanensis f. aureostriatus leaf extract showed significant antioxidant activities, DNA damage prevention ability, and effective effect on protecting organism from irradiation injury. Phenolic acid and flavonoids were the main active compounds. P-coumaric acid, ferulic acid, kurilensin A, siparunoside and tricin is the first time detected from P. kongosanensis f. aureostriatus. Furthermore, kurilensin A has never been reported in Phyllostachys bamboo so far, while siparunoside was first time reported in bamboo. Kurilensin A, with strong antioxidant activity and DNA damage prevention ability, was the key active compound in P. kongosanensis f. aureostriatus leaf contributing its antioxidant activity and radioprotection ability. In addition, P. kongosanensis f. aureostriatus with the advantage of fast growing and large leaf biomass have the potential to be developed as a kind of leafy bamboo grove.Results of this study expanded a new space for bamboo chemical elements and modern biological utilization of bamboo resources.更多还原