【摘要】：類胡蘿卜素因其廣泛的生理功能和難以替代的食用、藥用價值,被認為是最具發(fā)展?jié)摿Φ奶烊簧仡愂称诽砑觿┲�。本實驗室前期研究發(fā)現(xiàn)鎖擲酵母(Sporidiobolus pararoseus)菌株的發(fā)酵產(chǎn)物可能含有β-胡蘿卜素、γ-胡蘿卜素、圓酵母素(torulene)、紅酵母紅素(torularhodin)等多種類胡蘿卜素。其中圓酵母素和紅酵母紅素與番茄紅素化學(xué)結(jié)構(gòu)相似,可能具有抗氧化、抗癌等多種生物活性,但目前尚未見到國內(nèi)外相關(guān)報道。 因此,本研究首先采用有機溶劑提取得到色素混合溶液,后利用硅膠柱分離純化。純化的色素通過薄層層析、高效液相色譜及質(zhì)譜分析(LC-ESI-MS)對其種類進行鑒定。而后,采用DDPH(1,1-二苯基-2-三硝基苯肼)自由基清除體系、亞油酸抗氧化體系及過氧化氫誘導(dǎo)離體人肝癌細胞SK-HEP-1氧化損傷體系進行純化色素抗氧化性的評價;并通過體外培養(yǎng)細胞(人肝癌細胞SK-HEP-1)形態(tài)學(xué)觀察、細胞存活力實驗分析純化色素對體外培養(yǎng)細胞的作用,乳酸脫氫酶(LDH)檢測、細胞周期分析其作用機理。通過以上研究得到以下結(jié)果: (1)鎖擲酵母發(fā)酵產(chǎn)物中的色素通過硅膠柱層析可以得到有效分離,根據(jù)其色譜結(jié)果判定所得色素的性質(zhì)符合類胡蘿卜素的特性,圓酵母素和紅酵母紅素是鎖擲酵母發(fā)酵產(chǎn)物。 (2) DDPH自由基清除體系和亞油酸抗氧化體系結(jié)果表明,圓酵母素和紅酵母紅素均具有較強的體外抗氧化活性,且圓酵母素的抗氧化活性尤為顯著。過氧化氫誘導(dǎo)離體人肝癌細胞SK-HEP-1氧化損傷體系的結(jié)果表明實驗濃度范圍內(nèi),圓酵母素和紅酵母紅素并不單一表現(xiàn)出抗氧化修復(fù)的作用,高濃度條件下可能會出現(xiàn)協(xié)同損傷的現(xiàn)象。 (3)經(jīng)圓酵母素和紅酵母紅素處理的細胞可導(dǎo)致SK-HEP-1細胞發(fā)生皺縮、細胞顆粒增多等形態(tài)學(xué)改變。細胞存活力實驗顯示圓酵母素和紅酵母紅素可以顯著抑制SK-HEP-1細胞存活力,作用強度呈時間-劑量效應(yīng),并且紅酵母紅素的48 h的半抑制濃度為2.4μmol/L,顯著強于番茄紅素。 (4)圓酵母素和紅酵母紅素處理SK-HEP-1細胞后,均未觀測到LDH釋放增加的現(xiàn)象,說明該兩種類胡蘿卜素對細胞存活力的抑制和細胞毒性無關(guān)。流式分析結(jié)果顯示圓酵母素和紅酵母紅素都是通過抑制細胞周期,使SK-HEP-1細胞阻滯在G0/G1期達到抑制增殖的結(jié)果,細胞增殖指數(shù)明顯降低。 綜上所述,圓酵母素和紅酵母紅素是鎖擲酵母的發(fā)酵產(chǎn)物,具有較強的體外抗氧化活性,可通過阻滯細胞周期來抑制體外培養(yǎng)人肝癌細胞SK-HEP-1的增殖,且紅酵母紅素的抑制作用顯著強于番茄紅素。
[Abstract]:Carotenoids are considered to be one of the most potential natural pigment food additives because of their extensive physiological functions and irreplaceable edible and medicinal value. Previous studies in our laboratory found that the fermentation products of (Sporidiobolus pararoseus) strain may contain many kinds of carotene, such as 尾-carotene, gamma-carotene, circular yeast (torulene), red yeast lycopene (torularhodin) and so on. Among them, the chemical structure of circular yeast and red yeast lycopene is similar to that of lycopene, and may have many biological activities, such as antioxidation, anticancer and so on, but there are no reports at home and abroad. Therefore, in this study, the pigment mixed solution was extracted by organic solvent, and then separated and purified by silica gel column. The purified pigment was identified by thin layer chromatography, high performance liquid chromatography and mass spectrometry (LC-ESI-MS). Then, DDPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging system, linoleic acid antioxidant system and hydrogen peroxide-induced SK-HEP-1 oxidative damage system were used to evaluate the antioxidant activity of purified pigments. The morphological observation of human hepatoma cell line (SK-HEP-1) was carried out in vitro. The effect of purified pigment on the cultured cells was analyzed by cell viability test. Lactate dehydrogenase (LDH) (LDH) and cell cycle analysis were used to analyze the mechanism of action of the purified pigment on the cultured cells in vitro. The results are as follows: (1) the pigment of yeast fermentation product can be separated effectively by silica gel column chromatography, and the properties of the pigment can accord with the character of carotenoid, according to the chromatographic results, the results show that the pigment in yeast fermentation product can be separated effectively by silica gel column chromatography. Saccharomyces cerevisiae and Saccharomyces cerevisiae are the fermentation products of lock-throw yeast. (2) the results of DDPH free radical scavenging system and linoleic acid antioxidant system showed that both lycopene and lycopene had strong antioxidant activity in vitro, and the antioxidant activity of lycopene was especially significant. The results of oxidative damage system of human hepatocellular carcinoma cell line SK-HEP-1 induced by hydrogen peroxide showed that Saccharomyces cerevisiae and Saccharomyces cerevisiae did not have the effect of antioxidant repair in the range of experimental concentration. The phenomenon of synergistic damage may occur under the condition of high concentration. (3) the cells treated with Saccharomyces cerevisiae and Saccharomyces cerevisiae could cause morphological changes such as shrinkage and increase of cell particles in SK-HEP-1 cells. Cell viability test showed that lycopene and lycopene could significantly inhibit the viability of SK-HEP-1 cells in a time-dose dependent manner, and the semi-inhibitory concentration of lycopene was 2.4 渭 mol / L for 48 h. Significantly stronger than lycopene. (4) the increase of LDH release was not observed in SK-HEP-1 cells treated with circular yeast and mycopene, which indicated that the inhibitory effect of these two carotenoids on cell viability was not related to cytotoxicity. The results of flow analysis showed that both circular yeast and red yeast lycopene could inhibit the proliferation of SK-HEP-1 cells in G0/G1 phase by inhibiting the cell cycle, and the cell proliferation index was significantly decreased. In conclusion, Saccharomyces cerevisiae and Saccharomyces cerevisiae are the fermentation products of Saccharomyces cerevisiae, which have strong antioxidant activity in vitro and can inhibit the proliferation of human hepatocellular carcinoma cell line SK-HEP-1 by blocking the cell cycle. The inhibitory effect of Saccharomyces cerevisiae was significantly stronger than that of lycopene.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R151

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本文編號：2473718