【摘要】：樟樹主要產(chǎn)于我國長江以南地區(qū)。樟樹枝繁葉茂,樹形優(yōu)美,不僅能吸收硫、氟等有害氣體,還具有防塵、驅(qū)蟲的功效,因此樟樹常被用來美化環(huán)境、凈化空氣。樟樹葉、枝具有散寒、止痛、補火、助陽的功效,除此之外還具有治療各種痛癥的功效。樟樹葉中含有精油、多酚、黃酮、木脂素等天然活性物質(zhì)。目前對樟葉中的精油、多酚、黃酮類物質(zhì)研究的較多,但是對樟樹葉中木脂素類物質(zhì)的研究較少,本文是以樟樹葉為原料,優(yōu)化不同提取技術(shù)優(yōu)化樟樹葉中木脂素工藝條件,對粗提取物進行分離純化并利用高效液相法(HPLC)分析分離物質(zhì)的純度,利用對DPPH自由基、羥基自由基、超氧離子自由基的清除效果和還原力的測試判斷木脂素的抗氧化;通過對大腸桿菌、枯草芽孢桿菌、金黃色葡萄球菌、綠膿桿菌、沙門氏菌、黑曲霉、青霉的抑菌活性判斷木脂素的抑菌效果;以宮頸癌細胞(HeLa)為研究對象,通過MTS,FCM,電化學(xué)法考察木脂素的抗癌效果。結(jié)果如下:(1)利用傳統(tǒng)方法—乙醇回流法為基礎(chǔ)方法,它主要是考察單因素乙醇濃度、料液比、回流時間對樟樹葉中木脂素得率的影響,在單因素和正交試驗的基礎(chǔ)上優(yōu)化提取工藝,最終得到最佳的工藝水平。最佳水平為:乙醇濃度為100%,料液比為1:4(g/mL),回流時間為2 h。(2)利用微波輔助技術(shù)提取樟樹葉中木脂素類物質(zhì)。主要是通過單因素試驗和響應(yīng)面試驗對微波輔助提取工藝進行優(yōu)化。得出響應(yīng)面優(yōu)化的最佳工藝條件為:微波時間5 min、料液比1:26(g/mL)、乙醇濃度80%、微波溫度60℃。(3)利用超聲波輔助技術(shù)提取樟樹葉木脂素,結(jié)合單因素和響應(yīng)面試驗優(yōu)化提取工藝,結(jié)果表明在料液比1:22 g/mL、超聲時間3 min、乙醇濃度60%、提取時間78 min條件下,木脂素得率最大。(4)利用對DPPH自由基、羥基自由基、超氧離子自由基的清除效果和還原力的測試判斷粗木脂素類物質(zhì)的抗氧化性能。結(jié)果表明:木脂素具有較好的抗氧化活性,在此基礎(chǔ)上對粗木脂素進行分離純化。(5)通過大孔樹脂、硅膠、LH-20、ODS等柱色譜,薄層色譜(TLC)對樟樹葉中木脂素類物質(zhì)進行純化。分別利用紫外分光光度法(UV)、傅里葉紅外色譜(FT-IR)、質(zhì)譜(MS)、核磁共振(NMR)中的氫譜(1H-NMR),碳譜(13C-NMR)對其進行結(jié)構(gòu)鑒定。根據(jù)紫外區(qū)的最大吸收峰,紅外色譜不同波段的透射峰,質(zhì)譜數(shù)據(jù),核磁共振中的氫譜及碳譜中的吸收值最終確定樟樹葉中的3種木脂素,分別為松脂素-β-D-吡喃葡萄糖苷、松脂素、芝麻素。通過外標法采用高效液相法(HPLC)對分離出的3種物質(zhì)進行純度鑒定。(6)本章則通過肉湯稀釋法和紙片法測定從樟樹葉中分離到的松脂素對枯草芽孢桿菌和綠膿桿菌的抑制作用。在此基礎(chǔ)上利用光學(xué)顯微鏡(LM)、掃描電鏡(SEM)、生長曲線、細胞膜的通透性說明了松脂素抑制枯草芽孢桿菌和綠膿桿菌的機制。(7)利用MTS法檢測了不同濃度的松脂素對宮頸癌細胞(HeLa)和人正常上皮細胞(HUVEC)的抑制作用,發(fā)現(xiàn)松脂素以濃度-效應(yīng)的關(guān)系抑制HeLa細胞和HUVEC細胞的生長,并且對HeLa細胞的IC50為200 μg/mL。利用流式細胞術(shù)(FCM)和Hoechst 33258染色技術(shù)檢測了松脂素處理前后的HeLa細胞的凋亡情況和周期情況。(8)利用羧基化的碳納米管和金納米粒子溶液對玻碳電極進行修飾從而放大電化學(xué)信號。通過循環(huán)伏安法考察不同體積比羧基化的碳納米管和金納米粒子溶液對電化學(xué)信號的影響進行優(yōu)化,最終確定了碳納米管的體積與金納米粒子溶液的比例。利用循環(huán)伏安法(CV)和交流阻抗譜(EIS)分別對不同細胞濃度和不同松脂素濃度對宮頸癌細胞進行測定。與傳統(tǒng)方法相比較,電化學(xué)法快速簡便的測定松脂素對宮頸癌細胞的誘導(dǎo)凋亡作用,為快速測定宮頸癌提供了一個潛在的方法。
[Abstract]:The camphor tree is mainly located in the south of the Yangtze River. The camphor tree is luxuriant, the tree is beautiful, not only can absorb the harmful gas such as sulfur, fluorine and the like, but also has the functions of dust-proof and insect-expelling, so that the camphor tree is often used for beautifying the environment and purifying the air. The camphor tree leaves and branches have the effects of dispersing cold, relieving pain, tonifying fire, and promoting yang, and has the effects of treating various pain diseases. The camphor tree leaf contains natural active substances such as essential oil, polyphenolic, flavone, and lignan. At present, the research of essential oil, polyphenols and flavonoids in the leaves of Cinnamomum camphora has been studied, but the study of the lignan in the leaves of the camphor tree is less, this paper is to optimize the process conditions of the lignan in the leaves of the camphor tree by using the leaves of the camphor tree as the raw material and optimizing the different extraction techniques. the crude extract is separated and purified and the purity of the separated substance is analyzed by high-performance liquid-phase method (HPLC), The antibacterial activity of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Aspergillus niger and Paecilomyces was determined, and the anti-cancer effect of the lignan was studied by MTS, FCM and electrochemical method. The results are as follows: (1) The method is based on the traditional method of ethanol reflux, which is mainly used to study the effect of single factor ethanol concentration, liquid ratio and reflux time on the yield of the lignan in the leaves of the camphor tree, and the extraction process is optimized on the basis of the single factor and the orthogonal test. and finally the optimal process level is obtained. The best level is that the ethanol concentration is 100%, the ratio of the liquid to liquid is 1: 4 (g/ mL), and the reflux time is 2 h. (2) the lignan in the leaves of the camphor tree is extracted by the microwave-assisted technique. The microwave-assisted extraction process was optimized by single factor test and response surface test. The optimum conditions for the response surface optimization were: microwave time 5 min, material liquid ratio 1: 26 (g/ mL), ethanol concentration of 80%, and microwave temperature of 60 鈩，

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