以Caco-2細胞模型篩選具調節脂質吸收能力之中藥材與植物萃取物

Screening of traditional Chinese medicine and plant extracts with the ability to regulate the absorption of lipid by Caco-2 cells model

陳乃瑄(Nai-Hsuan Chen)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102983

摘要

高膽固醇血症（hypercholesterolemia）為血液中總膽固醇或低密度脂蛋白膽固醇（low density lipoprotein cholesterol）高於正常值，增加罹患動脈粥狀硬化（atherosclerosis）之風險。由於其症狀不易被察覺，因此患者往往等到罹患心血管相關疾病方始察覺。故在慢性惡化之病程中，透過改善日常生活習慣來達到減緩相關疾病發生為根本之預防方法。目前已知有多種天然食品成分具有保健之功效，若能透過其來達到預防或減緩高膽固醇血症發生的風險，將具有降低心血管疾病（cardiovascular diseases）及其相關併發症發生之潛力。本研究以腸道上皮細胞Caco-2細胞作為模型，篩選具影響脂質吸收能力之中藥材或植物萃取物。實驗分為三個部分，第一部分會對中藥材與植物，包括五味子（Schisandra chinensis, SC）、覆盆子（Rubus idaeus, RI）、山茱萸（Cornus officinalis, CO）、北蟲草子實體（Cordyceps militaris, CM）、北蟲草發酵殘餘物（C. militaris spent, CMS），以及台灣藜（Chenopodium formosanum Koidz, Djulis）進行萃取，並檢測萃取物中所含之生理活性成分（biologically active components）。第二部分為利用腸上皮細胞吸收實驗模型，篩選具降低膽固醇及三酸甘油酯（triglyceride）吸收能力之萃取物並找到其最低有效劑量，再以即時定量聚合酶連鎖反應（real-time polymerase chain reaction）分析細胞中膽固醇與三酸甘油酯代謝相關基因表現量變化。最後，針對具影響膽固醇與三酸甘油酯吸收能力之中藥材與植物萃取物進行指標成分（chemical marker）分析。結果顯示，五味子、覆盆子、山茱萸、北蟲草子實體、北蟲草發酵殘餘物及台灣藜所含之粗多醣（crude polysaccharide）分別為44.14 ± 3.38、196.18 ± 2.36、12.52 ± 0.13、73.86 ± 2.89、48.15 ± 4.46及29.40 ± 6.47 mg/ml；酚類化合物（phenolic compound）於五味子、覆盆子、山茱萸及台灣藜分別為6.90 ± 0.08、256.53 ± 3.90、17.12 ± 0.20及15.36 ± 0.14 mg/ml；黃酮類化合物（flavonoids）分別為4.56 ± 0.16、21.38 ± 1.82、0.78 ± 0.04及5.96 ± 0.42 mg/ml。第二部分，證實北蟲草、覆盆子與台灣藜萃取物分別在濃度2、2與1.5 mg/ml時，可影響細胞內之膽固醇與三酸甘油酯之吸收量，且植物萃取物之功效性可能與其黃酮類化合物含量有關。於膽固醇代謝相關基因表現層面，發現三種萃取物皆可透過抑制NPC1L1表達以達到降低膽固醇吸收。此外，植物萃取物可透過NPC1L1-SREBP2-LDLR路徑來達到回收低密度脂蛋白膽固醇。三酸甘油酯代謝基因層面，覆盆子與台灣藜萃取物可透過增加PPARα表達，減少細胞三酸甘油酯濃度。指標成分測定部分，北蟲草子實體及北蟲草發酵後殘餘物萃取物所含之蟲草素分別為13112 ± 276 μg/g及10839 ± 527 μg/g；台灣藜萃取物所含之芸香苷濃度為1650 ± 80 μg/g；覆盆子萃取物所含之鞣花酸濃度為56599 ± 9859 μg/g。綜上所述，本研究證實北蟲草子實體、覆盆子與台灣藜萃取物具影響腸上皮細胞膽固醇與三酸甘油酯代謝能力，未來可能作為降膽固醇相關保健食品之開發材料。

關鍵字

北蟲草 ； 台灣藜 ； 覆盆子 ； 高膽固醇血症 ； Caco-2 cell

並列摘要

Hypercholesterolemia was high level of total cholesterol and low-density lipoprotein cholesterol in plasma that was increase the risk of atherosclerosis. Because the symptoms of getting hypercholesterolemia are not easy to conscious, until suffering from cardiovascular relative diseases. Therefore, taking precaution by lifestyle to reduce the risk of suffering from cardiovascular diseases was essential. Currently, we know many of natural foods ingredients has the beneficial effect on health protection. If that can reduce the risk of suffering from hypercholesterolemia, it will have the potential to prevent cardiovascular disease. In this study, we screened of Chinese medicinal materials or plants, including Schisandra chinensi (SC), Rubus idaeus (RI), Cornus officinalis (CO), Cordyceps militaris (CM), C. militaris spent (CMS), Chenopodium formosanum Koidz (Djulis), with the ability of lipid absorption based on Caco-2 cell model. The experiment was divided into three parts. Firstly, we extracted these Chinese medicinal materials and plants, and detect the biologically active components in these extracts. Secondly, we selected the extracts with the ability of cholesterol and triglyceride absorption regulation and evaluating its minimum effective dose based on epithelial cell absorption experimental model. Then, we analyzed the gene expression of cholesterol and triglyceride absorption and metabolism relative gene by RT-PCR. Finally, we analyzed the marker component of the extracts with the ability of cholesterol and triglycerides absorption. The result shows that the content of crude polysaccharide was 44.14 ± 3.38, 196.18 ± 2.36, 12.52 ± 0.13, 73.86 ± 2.89, 48.15 ± 4.46, and 29.40 ± 6.47 mg/ml in SC, RI, CO, CM, CMS, and Djulis, respectively. The content of phenolic compounds was 6.90 ± 0.08, 256.53 ± 3.90, 17.12 ± 0.20, and 15.36 ± 0.14 mg/ml in SC, RI, CO, and Djulis, respectively. And the content of flavonoids was 4.56 ± 0.16, 21.38 ± 1.82, 0.78 ± 0.04, and 5.96 ± 0.42 mg/ml in SC, RI, CO, and Djulis, respectively. The second part confirmed that CM, RI, and Djulis extracts had ability to affect the cholesterol and triglyceride absorption at concentrations of 2 mg/ml, 2 mg/ml and 1.5 mg/ml, respectively. Moreover, the activity component of plants extracts would probably relate to the content of flavonoids. In gene expression of cholesterol metabolism level, we found that three extracts could reduce the cholesterol absorption through inhibiting the expression of NPC1L1. In addition, plant extracts could affect the LDL-C recovery through NPC1L1-SREBP2-LDLR pathway. In gene expression of triglyceride metabolism level, we found that RI and Djulis could affect the triglyceride absorption through promoting the expression of PPARα. In the part of chemical marker analysis, the content of cordycepin in extracts of CM and CMS was 13112 ± 276 and 10839 ± 527 μg/g, respectively. The content of rutin in extract of Djulis was 1650 ± 80 μg/g. And the content of ellgic acid in extract of RI was 56599 ± 9859 μg/g. In summary, this study confirmed that CM, RI and Djulis extracts have the ability to regulate lipid metabolisms in intestinal epithelial cells, and the extracts have potential to development into health foods with reducing lipid absorption in the future.

並列關鍵字

Cordyceps militaris ； Chenopodium formosanum Koidz ； Rubus idaeus ； hypercholesterolemia ； Caco-2 cell

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