介質研磨枸杞之製備及其細胞保護功效

Abstract

奈米科技已廣泛應用於不同產業，其應用於食品產業於正迅速發展。本研究以枸杞為研究食材，強調全食材之攝取，期望製備出品質穩定且具有保健功效的介質研磨枸杞。本研究首先利用介質研磨技術製備奈米/次微米尺寸之介質研磨枸杞，介質研磨枸杞之粒數平均粒徑顯著下降至100 nm，其表面負電增加，並且展現較佳的粒子穩定性。介質研磨枸杞中水溶性膳食纖維、粗多醣、總類胡蘿蔔素及beta-胡蘿蔔素之萃取率顯著增加。藉由細胞安全性評估發現，介質研磨枸杞不影響大鼠小腸上皮細胞(IEC-6)、人類結腸腺癌細胞(Caco-2)及小鼠初代脾臟細胞之正常生長。利用SD大鼠組織分布觀察枸杞之生物利用率，發現於餵食研磨枸杞30天後，其中血清、肝臟及脾臟玉米黃素(zeaxanthin)濃度皆顯著增加。 利用oligomicroarray篩選出小鼠餵食細碎或研磨枸杞後，脾臟組織中表現量具有顯著變化的基因。發現餵食介質研磨枸杞之小鼠脾臟中與細胞生存相關的TNF-alpha、NF-kappaB 及 Bcl-2 之mRNA表現量顯著增加，而與細胞凋亡相關之APAF-1和Caspase-3之mRNA表現量顯著降低，此mRNA表現量利用及時定量PCR驗證後結果一致。深入探討細胞保護活性及機制發現，研磨枸杞顯著降低由camptothecin 所誘發之初代脾臟細胞凋亡及細胞caspase-3活性。經細胞增生試驗顯示介質研磨枸杞並未造成細胞異常增生。利用西方墨點法分析，發現介質研磨枸杞顯著增加小鼠初代脾臟細胞TNF-alpha、NF-kappaB及Bcl-2之表現量，而caspase 3及Apaf-1之表現量顯著降低。推測枸杞之細胞保護機制由TNF-alpha及轉錄因子NF-kappaB相互配合，增加下游抗凋亡蛋白Bcl-2之表現量，進而抑制促進凋亡之caspase3及Apaf-1之表現。 深入研究神經細胞保護及機制發現，細碎及介質研磨枸杞皆顯著減少因缺乏血清所造成的PC12細胞凋亡。經細碎及介質研磨枸杞預處理顯著回復因添加NF-kappaB專一性oligonucleotide所降低的細胞生存率。推測枸杞樣品可能提高NF-kappaB的表現量而減少因缺血清所造成的PC12細胞凋亡。利用西方墨點法分析，發現介質研磨枸杞顯著提高NF-kappaBp50及p65的表現量，顯示枸杞藉由活化NF-kappaB維持神經細胞生存活性，展現神經細胞保護功效。 綜合結果顯示，枸杞經過介質研磨微細化，粒徑顯著下降，表面積增加，表面負電及粒子穩定性增加。增加有效成份之萃取率，提升動物體對有效成分zeaxanthin之生物可利用性，提高細胞存活相關基因的表現，主要的調節機制牽涉apoptosis and survival TNFR1 signaling pathway，進而增進細胞保護及神經保護之活性。

Nanomaterials have been applied in various industries, in which food nanomaterials are rapidly developed and employed worldwide. In this study, we use goji (Lycium barbarum) as a material, focusing on the intake of whole materials, and exploring the potential of development of health food product. Media milling was employed to prepare nano/submicron scaled goji. The number average diameter of goji was reduced to 100 nm after 90-min media milling. The higher surface negative charge might be attributed to the better particle dispersion and stability. The extraction efficacy of soluble dietary fiber, crude polysaccharides, total carotenoids and beta-carotene were elevated by size reduction. For the safety concern, we use cell culturing test to observe the cell viability. The cell viabilities of rat normal small intestinal cell line IEC-6, human colon adenocarcinoma cell line Caco-2 and primary mouse splenocytes did not directly influenced by media milling. The bioavailability of zeaxanthin was revealed by biodistribution of SD rats. Intake of media milled goji waa resulted in the increase of zeaxanthin concentration in serum, livers and spleens. Oligomicroarray and QPCR were revealed that three genes related to cell survival, TNF-alpha, NF-kappaB and Bcl-2 were up regulated, and two genes related to pro-apoptosis, APAF-1 and Caspase3 were down regulated by goji.Western blotting results also confirmed the effects of regulation on protein expression levels of those genes. Cytoprotective activities and mechanisms were further investigated in vitro. Media-milled goji significantly reduced camptothecin-induced cell death and caspase-3 activity of mouse primary splenocytes, nevertheless, it did not induce uncontrolled cell proliferation. It suggested that the coordination between TNF-αand NF-κB induce downstream anti-apoptotic protein Bcl-2 expression, further down regulated pro-apoptotic protein Caspase3 and Apaf-1 expression. For the neuroprotective effects, both samples dose dependently prevented PC12 cells from apoptosis caused by serum deprivation. NF-kappaB acts as an importantsurvival factor was revealed by NF-kappaB specific double strand oligonucleotidetreatment and Western blotting. Serum-deprived PC12 cell viability and NF-kappaB p50/p65protein expression were significantly elevated by media-milled goji. Activation of NF-kappaB by media-milled goji seemed to beresulted in better cell viabilities. In conclusion, reduced particle size, large surface area, more negative charge and better particle stability of media-milled goji were helpful for extraction of bioactive compounds and zeaxanthin bioavailability, further be attributed to the enhanced cytoprotective and neuroprotective activities.