第一部分: 利用Dabsylation及高效液相層析系統分析茶的茶胺酸及伽瑪胺基丁酸含量; 第二部分: 龍葵葉子水萃物促使AU565乳癌細胞進行自噬作用

Part I Analysis of Theanine and GABA contents in Teas (from Camellia sinensis L. and Apocynum venetum L.) by Dabsylation and HPLC system; PartII The water extract of Solanum nigrum Linn leaf induced autophagy in AU565 breast cancer cells

許凱揚(Kai-Yang Syu)

指導教授 ： 林仁混

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.00034

摘要

第一部分
Dabsyl chloride (dimethylaminoazobenzene sulfonyl chloride)，在1975年由本實驗室林仁混老師發展而成，可與氨基(amino)及胺類化合物(amine)反應(Dabsylaiton)，被廣泛的使用於胺基酸的分析，在本實驗裡，我們藉由Dabsylation反應及高效液相層析(HPLC)系統在吸光值425nm波長下，分析不同茶，包括綠茶、紅茶、烏龍茶、普洱茶、珈碼茶 (GABA tea) 及羅布麻茶 (luobuma tea) 的茶胺酸(Theanine)、珈瑪氨基丁酸(GABA; γ-aminobutyric acid)及其他胺基酸。在我們的分析中，發現同種茶的茶胺酸不盡相同，產地及製作過程等可能影響此差異，高山茶葉似乎含有較高量的茶胺酸，不論是綠茶還是烏龍茶；普洱生茶(未堆渥發酵)的茶胺酸比普洱熟茶(經堆渥發酵)的茶胺酸多。另外，珈瑪氨基丁酸在一般茶的含量通常不高，除了珈瑪茶，我們發現一些羅布麻茶的珈瑪氨基丁酸含量很高，羅布麻茶含有高量量的珈瑪氨基丁酸是第一次被發現的。

第二部分
Solanum nigrum Linn,中文又稱為龍葵、苦葵、黑甜仔菜，為茄科茄屬植物，可在亞洲溫帶季候區之田野中發現，一般傳統中草藥描述其性味苦寒有小毒，具利水消腫、清熱解毒及保肝之功效，然而龍葵的藥用機轉乃須更進一步的探討與研究。在我們實驗室，我們使用龍葵葉子的水萃物，以MTT比較龍葵對不同細胞的效應，包括肝癌細胞、乳癌細胞、及纖維組織母細胞，發現AU565乳癌細胞較具敏感性。龍葵水葉子萃物引發AU565進行自噬作用而非細胞凋亡，其作用機轉似乎與Akt或AMPK路徑的調控無關，低濃度(< 25 μg/ml)的龍葵葉子水萃物引起之自噬作用並無法使得細胞死亡，可能是因為自噬作用被後期增加之p-Akt所抑制，高濃度(> 100 μg/ml)之龍葵葉子水萃物可抑制此增高之p-Akt進而促使細胞死亡，這些結果再一次確認了Akt對於AU565癌細胞之重要，而龍葵水萃物對於治療AＵ565相關之癌症，仍需進一步評估。

關鍵字

茶胺酸 ； 伽瑪胺基丁酸 ； 茶 ； 龍葵 ； 自噬作用 ； 乳癌

並列摘要

Part I
Dabsyl chloride (dimethylaminoazobenzene sulfonyl chloride), a useful chromophoric labeling reagent for amino acids and amines, was developed in our laboratory in 1975. Although several methods have been developed to determine various types of amino acids, a quick and easy method of determining theanine, GABA and other amino acids has not been developed in one HPLC system. Here, we analyze the free amino acid contents of theanine and GABA in different teas (green tea, black tea, oolong tea, Pu-erh tea, GABA tea, and luobuma tea) with a dabsylation and reverse phase high performance liquid chromatography (HPLC) system coupled with a detector at 425nm absorbance. In this system, our data suggests that different teas may be recognized by their various theanine contents. The high theanine content of high mountain tea was observed in both green tea and oolong tea. Furthermore, the raw (natural fermented) Pu-erh tea contained more theanine than ripe (wet fermented) Pu-erh tea. And the GABA contents in normal teas were significantly lower than that in Luobuma tea and GABA tea.

Part II
Solanum nigrum Linn belong to Solanaceae is a plant widely grows in south Asia and has been used in traditional folk medicine. It is believed to have many biology activities including antipyretic, diuretic, anti-cancer, and hepatoprotective effect. However, the exactally mechanism of such effects needed further research. In our lab, we used the water extract of Solanum nigrum Linn leaf (SN leaf) to compare the response of different cells, including liver cancer, breast cancer, and fibroblast. The results showed that AU565 bresat cancer cells were more sensitive to the extract. SN leaf induced autophagy but not apoptosis in AU565. The induced autophagy seemed not related to Akt or AMPK pathway, but the exactly mechanism was still unknown. Low dose (< 25 μg/ml) of SN leaf induced autophagy would not induce cell death because of the raising of p-Akt to inhibit autohpagy. Higher dose (> 100 μg/ml) of SN leaf could inhibit the level of p-Akt and cause the cell death. These confirmed that Akt is the key survival factor for AU565, and the using of SN leaf for the treatment of AU565 related cancers were needed further evaluation.

並列關鍵字

theanine ； GABA ； tea ； dabsylation ； Solanum nigrum Linn ； autophagy ； breast cancer

參考文獻

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