天然物成分之基因毒性評估：

天然物成分之基因毒性評估： Podophyllin 誘發基 因損傷機制之探討及其致突變成分分析

 中草藥之使用已有數千年歷史，近年又隨著替代醫學興起，銷售量日益增加，為維護民眾健康，我國將之列屬藥品管理。由於科技的發展，改變了人們食 用這些中草藥的方式，不再以原始植物型態攝食，而是將其萃取濃縮成粉末或碇劑形式食用，大大的提升了攝入劑量。導致過去傳統的食用經驗與所謂的 安全概念已不適用於人類現今服用中草藥的形式。對於這些常見於中草藥中之成份，其毒性作用，有需要重新再評估。而在所有可能的致毒機轉中，基因 毒性是人類遲至 19 世紀才開始注意以及有能力去研究的領域，對於慣用過去食用經驗當成安全性考量的中草藥而言，其未經測試的基因毒性作用，更是 一大隱憂。基於天然化學物質在致基因突變研究上的了解有限，因此將其定為本論文探討的重點。本論文的主要目的欲建立一個低成本高效率之基因毒性 篩選模式，進行廣泛且數量大的篩選，之後再針對所篩選出之疑似致突變物，進行進一步的機制探討及確認。在過去幾年的篩選工作總共完成了 36 種中 藥成份之研究，其中發現了幾種疑似致基因突變物，因此將本論文延申至第二及第三部份。總結，本論文內容可分為三部份， Part I ，首先以體外微生物 基因毒性試驗法 (Ames Test ; 安姆氏試驗 ) ，篩選缺乏文獻佐證其安全性之天然物成分。這些篩選之成分，若依其化學結構分類，涵蓋黃酮類、有機酸 ( 酚 ) 類、香豆素類、萜類、生物鹼類及配醣體類等。試驗之成分包括 podophylli 等 36 種成分。以三株沙門氏菌 (TA98 、 TA100 及 TA102) 進行測試，於 添加或不添加代謝活化酵素條件下，測試細菌之逆突變作用。結果顯示， quercetin 及 podophyllin 以濃度－反應關係，呈現有意義地增加逆突變菌落數目

，因此初步推測 quercetin 及 podophyllin 具有潛在基因毒性。由於 podophyllin 為目前隨處可購得之產品，倘民眾無意識地、大量地或長期使用，恐對民眾 健康產生影響。因此，選定 podophyllin 作為第二部分基因毒性之研究與探討。

由於 podophyllin 本身是一種植物萃取物，成分複雜，欲了解基因毒性則需先進行成分分析，始能探討何種成分造成其基因毒性之表現。在論文第二部份 Part II 包含兩大主題，一為 podophyllin 之成分分析 (Part II-A) ，另外則為 podophyllin 致基因突變作用機制之探討 (Part II-B) 。首先於第一部份 (Part II- A) ，先行開發一種高效液相層析串聯式質譜法，以應用於其成分分析。經由文獻搜尋， Podophyllin 主要含 podophyllotoxin 、 quercetin 及 kaempferol 等三 種成分。進行含量測定前，需先確認其所含成分，定量分析前預先以上述三成分建立質譜資料庫，再經由質譜比對，確認含以上三種成分。續以多重離子 監控 (Multiple Reacting Monitoring ， MRM) 模式，選擇最佳的子代離子，進行成分之定量分析。經由檢量線分析，本試驗所使用之 podophyllin 含 podophy llotoxin 、 quercetin 及 kaempferol 分別占 podophyllin 之 31.2% 、 1.8% 及 3.2% 。本研究 Part II-B 部分則利用前述結果進行 podophyllin 及其成分之基因毒 性研究，目的藉以釐清 podophyllin 導致的基因毒性作用是否來自已知具「體外基因毒性作用」之 quercetin 或是 kaempferol 。試驗方法包括體外微生物基 因毒性、哺乳動物體外染色體變異試驗及動物體內微核分析。此三種基因毒性方法測試，均顯示 Podophyllin 具基因毒性，至於 quercetin 及 kaempferol 以 相對於 podophyllin 中之含量，只於 Ames test ，顯示致微生物逆突變作用，特別是 TA98 菌株於代謝活化酵素存在下有極強之基因毒性，但此致突變性並 沒有出現在染色體異常以及活體微核試驗。顯示 podophyllin 所導致的基因毒性作用並非來自 quercetin 或是 kaempferol ，這樣的發現也證實了有另外存在 於 podophyllin 內之化合物造成 podophyllin 之作用，也因此提高了使用 podophyllin 之安全疑慮。為探討造成 podophyllin 之基因毒性機制，我們也針對氧 化活性物質 (Reactive Oxygen Species ， ROS) 進行偵測，發現 podophyllin 可於 CHO 細胞誘導 ROS 之產生，而 ROS 之產生已公認與基因毒性有關。當以 同樣的試驗測試 3 種成份時，則有不同之表現。 Quercetin 或是 kaempferol 與 podophyllotoxin 皆不誘發 ROS 生成。顯示了 podophyllin 所導致的 ROS 與三 大主成分皆無關。

另外 podophyllin 之主成分 podophyllotoxin ，雖然於離體基因毒性試驗沒有作用，但於動物體內微核分析顯示，意外的發現具濃度關係的增加微核數目。

因此我們的論文在第三部份則持續探討 podophyllin 的藥理及毒理特性，在藥理作用方面，我們發現以螢光染劑 (propidium iodide ， PI) 進行 DNA 含量 ( 細 胞週期 ) 分析，發現 podophyllin 可以造成細胞凋亡、並使細胞停於 G2/M 期。細胞凋亡代表 Podophyllin 造成 DNA 傷害， DNA 傷害可能與 podophyllin 誘 導微核生成或誘使染色體變異有關。其中之 quercetin 或是 kaempferol 亦不影響 cell cycle 之表現。但是 podophyllotoxin 卻明顯的表現出與 podophyllin 相同 之調節 cell cycle 作用。在基因毒性作用方面，我們確認 podophyllotoxin 可能為一種 promutagen, 可於動物體內經代謝後產生致基因突變作用。

綜合以上之結果，我們結論，經過多達 36 種中草藥之篩選之後，發現 podophyllin 這個已被人類經常使用的中草藥成分在我們所測試的劑量下具有致基因 突變作用，我們確定此作用並非來自 quercetin 或是 kaempferol ，雖然另一成分 podophyllotoxin 於動物體內微核試驗表現出與 podophyllin 相似的結果， 但 我們仍舊無法以此確定 podophyllin 所導致的基因毒性來自 podophyllotoxin ，因為在離體動物試驗中顯示有另外的物質貢獻了 podophyllin 所造成的微生物 逆突變作用。我們也藉著本論文的完成，建立了研究含多種化合物之中草藥之基因毒性試驗模式，除了證實其致突變作用之外，也進一步的尋找其可替代 且更安全之物質。期待這樣的研究方式能廣及所有的未測試物質，以保障人民的用藥安全。

Genotoxicity evaluation of natural products : Mech anism of podophyllin induced genotoxicity and the

chemical analysis of podophyllin

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Herbal medicines have been used for more than thousands of years. The market sales increase gradually as the alternative medicine spring up. Herbal medicines are regulated as medicine in Taiwan. Due to the technology development, herbal medicines are taken not only the original type but also extracted powder or tablet prepared from herbal medicines, hence, the intak e amounts by human are augmented, the past traditional experience of intake and the concept of safety is not suitable for modern herbal medicine application on human. It is necessary to reevaluate the toxicity from the constituents of herbal medicine commonly used. Among the possible mechanism of intoxication, genotoxicity is the newly developed toxicity field, and it draws human’s attention as well as is capable of conducting on the research till the nineteenth century. It’s a secret worry for lacking of detailed genotoxicty data from herbal medicine bu t considered as safety from the intake experience. As the understanding on mutagenicity of natural products is confined, the thesis focuses on this field. The aim of the thesis is to establis h a low-cost and highly efficient screening mode, and applied to screen huge amounts of samples extensively, following the screening assay, the suspected mutagen is reconfirmed its gen otoxic effect and then investigates on the genotoxicity mechanism. Genotoxic screening on thirty-six natural compounds have been finished in the past several years. Some of them are su spected as mutagens, hence, we extend the related researches on parts two and three. Shortly, the thesis consists of three parts, Part I, First screening tests of the natural products, which is short of genotoxicity to support its safety are conducted by in vitro microbial genotoxicity (Ames Test). The chemicals by chemical structures classification include flavonoids, organic ac ids (phenols), coumarins, terpenoids, alkaloids and glycosides. The tested compound are as follows: allantoin, amygdalin, asarone, asiaticoside, baicalein, baicalin, caffeic acid, cinnamic acid, coumarin, costunolide, daidzein, digitoxin, 6,7-dimethoxycoumarin, ??-escin, ferulic acid, furolic acid, gentiopicroside, 18??-glycyrrhetic acid, hesperidine, imperatorine, isorhamne tin, kaempferol, naringin, neohesperidine, paeonol, podophyllin, podophyllotoxin, puerarin, quercetin, rutin, silymarin, silybin, strychnine, umbelliferone, wogonin and yohimbine. Salmo nella typhrium Strains of TA98, TA100 and TA102 were selected to perform the test, with and without the metabolic enzyme, observed the microbial mutagenicity. Quercetin and podoph yllin-induced revertants show concentration-response relationship, suggesting quercetin and podophyllin showed strong mutagenic effect potentially in the preliminary test. As podophyll in is easily obtained, the genotoxic activity needs to be cautioned. Hence, podophyllin was selected as the role in the part two.

As a mixture of podophyllin, the true contributor for genotoxicity has not been defined; it needs to verify the constituents. The part two consists of two subjects, one describes the chemic al analysis of podophyllin (Part II-A) and the other describes the investigation on genotoxic mechanism of podophyllin. An LC/MS/MS method was developed to analyze podophyllin. T he identification of constituent was conducted on daughter ion scanning mode, and the determination was conducted on the multiple reacting monitoring mode. The quantitative results fr om LC/MS/MS show the percentage of podophyllotoxin, kaempferol and quercetin in podophyllin were 31.2, 3.2 and 1.8%. Following the quantitative results, each constituent was used to evaluate the genotoxic potential in order to clarify the podophyllin-induced genotoxicity, whether the mutagenic effect is from known mutagenic effects of quercetin and kaempferol or not (Part II-B). The evaluation method for genotoxicity consists of microbial genotoxicty test and mammal chromosome aberration test in vitro as well as mammal micronucleus formatio n in vivo. Podophyllin showed positive reaction in Ames test, chromosome test and in vivo micronucleous test, but quercetin and kaempferol related to the contents in podophyllin used o nly showed genotoxicity in Ames test, especially, in the presence of metabolic activation systems in the strain TA98, but not in chromosome test and in vivo micronucleous test. From the above result, quercetin and kaempferol-induced mutagenic effect were excluded. On the other hand, this finding indicates that another compounds existing in podophyllin induce genotox icity, and than raises the doubt about the safety of podophyllin application. In order to investigate the genotoxic mechanism of podophyllin, reactive oxygen species (ROS) was also carrie d out. The ROS production assay showed that podophyllin increases the DCF fluorescenc in CHO cells, implying that ROS production was induced by podophyllin. The ROS production is accepted as related to genotoxicity. Different results are from three constituents by the same ROS assay. Quercetin or podophyllotoxin or kaempferol do not induce ROS production. Th e results showed that there is no relationship between podophyllin and its three separate components on the induction of ROS production by podophyllin

Besides, podophyllotoxin, the major constituent of podophyllin, do not display in vitro genotoxicty, but show the increase on the incidence of micronucleus formation by concentration-d ependently. Hence, we continuously investigate the pharmacological and toxicological properties of podophyllin. In the pharmacological study, podophyllin induced apoptosis, and result ed in G2/M arrest by propidium iodide based on DNA analysis (cell cycle). Apoptosis represents DNA damage is from podophyllin, and DNA damage might be result in micronucleus for mation or chromosome aberration. Among them, quercetin or kaempferol do not effect on cell cycle expression, but podophyllotoxin shows the same cell cycle modulating effects. We su ggest that podophyllotoxin might be as a promutagen and induce gene mutation by the metabolism in vivo

Collecting the above results, podophyllin, the commonly used herbal medicine, showed mutagenicty in the mutagenic screening work among thirty-six compounds, and the mutagenic eff

ect is not from quercetin or kaempferol. We are unable to confirm that the podophyllin-induced genotoxicity is from podophyllotoxin even though which shows the increase on micronucl

eus formation, other constituents contribute the genotoxicity in vitro microbial mutagenicity study. We establish the genotoxic-screening mode for herbal medicine by the accomplishmen

t of the thesis. In addition to verify its mutagenicty, find more safe and alternative substances in advance. We wish the research could expend to other un-testing substances in order to pro

tect the medication safety.