林紅燕，王煊，何聰，周紫玲，楊旻愷，文鐘靈，韓洪葦，陸桂華，戚金亮，楊永華

中藥植物紫草天然產(chǎn)物的生物合成及其功能研究進(jìn)展

林紅燕，王煊，何聰，周紫玲，楊旻愷，文鐘靈，韓洪葦，陸桂華，戚金亮，楊永華

南京大學(xué)醫(yī)藥生物技術(shù)國(guó)家重點(diǎn)實(shí)驗(yàn)室，植物分子生物學(xué)研究所，生命科學(xué)學(xué)院，南京 210023

紫草為我國(guó)傳統(tǒng)的重要藥用植物資源，其根部代謝產(chǎn)生的紫紅色萘醌類天然產(chǎn)物—紫草素及其衍生物，臨床上常被用于治療瘡瘍和皮膚炎癥。數(shù)十年來(lái)，紫草因具高效的多重生物活性、藥理作用、良好的臨床療效、較高的利用價(jià)值，引起了國(guó)內(nèi)外研究者的重視與關(guān)注，正由于此種原因，其野生植物種質(zhì)資源常遭到大量采挖，生長(zhǎng)環(huán)境受到嚴(yán)重威脅。隨著植物天然產(chǎn)物的生物合成、分子代謝及其生物技術(shù)的發(fā)展，藥用植物天然產(chǎn)物生物活性功能與藥理作用研究手段的不斷創(chuàng)新，紫草的生物合成途徑和相關(guān)調(diào)控基因的研究取得了顯著的進(jìn)展，紫草素藥理作用及其機(jī)制得到深入闡明或解析，極大地推進(jìn)了紫草素的基礎(chǔ)性研究及其臨床應(yīng)用開(kāi)發(fā)的進(jìn)程。本文從紫草分類、紫草素的結(jié)構(gòu)與組成及其生物合成途徑、調(diào)控紫草素生物合成代謝的功能相關(guān)基因以及紫草素生物活性與藥理功能等方面綜述了相關(guān)研究進(jìn)展，并對(duì)未來(lái)可能的發(fā)展趨勢(shì)進(jìn)行了展望，以期為促進(jìn)我國(guó)重要中藥材源的藥用天然產(chǎn)物的深度挖掘與開(kāi)發(fā)提供有益參考，推動(dòng)我國(guó)傳統(tǒng)中藥學(xué)的現(xiàn)代化發(fā)展。

紫草；紫草素；生物合成；基因調(diào)控；藥理活性

紫草(Zi Cao)是我國(guó)傳統(tǒng)中藥材，始載于《神農(nóng)本草經(jīng)》，列為中品，曰：“紫草，味苦、寒，主心腹邪氣五疸，補(bǔ)中益氣，利九竅，通水道，一名紫丹，一名紫芙，生山谷”?！吨腥A人民共和國(guó)藥典》(2020年版)收載的以干燥根為中藥材的紫草來(lái)源于紫草科軟紫草屬植物—新疆紫草((Royle)Johnst)或內(nèi)蒙紫草(Bunge)；2000年及其之前的藥典版本中則收載了紫草科紫草屬植物–紫草(Sieb. et Zucc.)等物種。這些紫草物種的根部均含有紫紅色的萘醌類藥用天然產(chǎn)物—紫草素及其衍生物(以下簡(jiǎn)稱紫草素)，具有抗菌、消炎、活血等功效，且可應(yīng)用于食品、高級(jí)化妝品、日化產(chǎn)品與塑料制品的著色等。近年來(lái)，國(guó)內(nèi)外的研究發(fā)現(xiàn)紫草素不僅具有抗菌消炎、抑制人類免疫缺陷病毒(human immu-nodeficiency virus, HIV)等多種藥理活性，還可通過(guò)活化Caspase-3和抑制拓?fù)洚悩?gòu)酶活性來(lái)誘導(dǎo)癌細(xì)胞凋亡，是繼喜樹(shù)堿、紫杉醇之后又一類極具潛力的天然抗腫瘤藥物。本文就紫草分類、紫草素結(jié)構(gòu)、生物合成及其基因調(diào)控以及紫草素生物活性與藥理功能等研究進(jìn)展，進(jìn)行了較為全面的概述，期望加強(qiáng)我國(guó)紫草素的基礎(chǔ)性研究及其應(yīng)用開(kāi)發(fā)。

1 紫草的分類

按照《中國(guó)植物志》第64(2)卷(1989)及其英文修訂版“”·Vol.16(1995)的形態(tài)分類系統(tǒng)，紫草科()約包含100多個(gè)屬，2000多個(gè)物種，廣泛分布于世界各地，以歐洲地中海區(qū)為分布中心[1,2]。目前，紫草科主要被劃分為紫草亞科(Subfam.)、天芥菜亞科(Subfam.)、破布木亞科(Subfam.)、厚殼樹(shù)亞科(Subfam.)這四個(gè)亞科，其中紫草亞科為其主要類群。紫草亞科中的紫草族(Trib.)主要包含了紫草屬()、軟紫草屬()、滇紫草屬()、藍(lán)薊屬()以及紫筒草屬()等5屬。其中，紫草屬與軟紫草屬、滇紫草屬，可能具有較近的親緣關(guān)系；按花器官分類，滇紫草屬可能處于較高的進(jìn)化地位，其次是軟紫草屬，而紫草屬則處于較原始狀態(tài)。上述5屬植物共有約50個(gè)物種，根據(jù)形態(tài)分類系統(tǒng)，廣義上可合并為紫草族，狹義上又可拆分為紫草屬與擬紫草屬()[3]。

2 紫草素的結(jié)構(gòu)、組成及其生物合成途徑

2.1 紫草素結(jié)構(gòu)與組成

“紫草”中藥材主要特指能入藥或做藥的“紫草科”植物富含紫草寧及其衍生物的紫紅色根，包括軟紫草屬的新疆紫草、內(nèi)蒙紫草，以及紫草屬的紫草和滇紫草屬的滇紫草等，根據(jù)其根部主要藥用成分的含量差異，新疆紫草和內(nèi)蒙紫草的根主要為藥用，紫草和滇紫草等可替代入藥，也被廣泛用做染料[4~6]。紫草素是一類萘醌化合物，其母核為5, 8-二羥基-1, 4-萘醌(5, 8-dihydroxy-1, 4-naphthoquinone)，并具異己烯基側(cè)鏈。根據(jù)其旋光性不同，紫草素類化合物被分為兩種旋光異構(gòu)體，即左旋紫草素(阿卡寧，-型，alkannin)與右旋紫草素(紫草寧，-型，shikonin) (圖1)[7~10]。

2.2 紫草素的生物合成途徑

紫草素的生物合成以產(chǎn)生香葉基–對(duì)羥基苯甲酸(m-geranyl-p-hydroxybenzoic acid, GBA或GHB)為節(jié)點(diǎn)，可分為上游部分與核心部分；上游部分包含了兩個(gè)途徑，第一個(gè)是由萜類骨架途徑(terpe-noid backbone biosynthesis)合成香葉酯焦磷酸(geranyl pyrophosphate, GPP)，第二個(gè)是由苯丙素途徑(phenylpropanoid biosynthesis, PP)產(chǎn)生對(duì)羥基苯甲酸(4-hydroxybenzoate acid, PHB) (圖2)[7~10]。

在生物體中，萜類前體骨架途徑包括了兩個(gè)途徑，即細(xì)胞質(zhì)中的甲羥戊酸代謝途徑(mevalonate pathway, MVA)和質(zhì)體中的去氧木酮糖途徑(2-C- methyl-D-erythritol-4-phosphate pathway, MEP)[11~14]。在紫草素生物合成的研究中，Gaisser 等[15]發(fā)現(xiàn)抑制紫草MVA途徑中的限速酶甲戊二羥酸單酰輔酶A還原酶(hydroxymethylglutaryl-coenzyme A reductase, HMGR)活性，幾乎能夠完全抑制紫草素的生物合成。由此，研究者推斷紫草的紫草素合成途徑中GPP主要來(lái)源于MVA途徑。但是，近年Singh等[16]發(fā)現(xiàn)，當(dāng)軟紫草MEP途徑中的限速酶5-磷酸–去氧木酮糖還原酶(5-phosphoric acid-deoxyxylulose reductase, DXR)活性被抑制后，軟紫草中的紫草素合成同樣受到抑制。由此可見(jiàn)，紫草素生物合成過(guò)程中，MVA和MEP這兩個(gè)途徑之間的關(guān)系還有待進(jìn)一步研究和確定。

圖1 紫草中的紫草素類天然產(chǎn)物

根據(jù)參考文獻(xiàn)[9]修改繪制。A：紫草寧類；B：阿卡寧類。R為取代基團(tuán)；1：紫草素；2：去氧紫草素；3：乙酰紫草素；4：ɑ-甲基-正丁酰紫草素；5：異丁酰紫草素；6：異戊酰紫草素；7：β-羥基異戊酰紫草素；8：β,β?-二甲基丙烯酰紫草素；此外，4與6又屬于同分異構(gòu)體。

PHB是紫草素生物合成的另一重要前體，在生物體中，其可由兩個(gè)途徑而來(lái)，分別是PP與莽草酸途徑(Shikimate pathway)[17,18]。研究發(fā)現(xiàn)，莽草酸途徑不僅可直接合成分支酸，經(jīng)由分支酸-丙酮酸合成酶(chorismate-pyruvate lyase, CPL或UbiC)直接合成PHB，還是PP的上游途徑，能夠?yàn)镻P途徑提供苯丙氨酸(Phenylalanine)。然而，PP中4-香豆酸(4-Coumarate)經(jīng)4-香豆酸輔酶A連接酶(4-coumarate: CoA ligase, 4CL)催化合成4-Coumaroyl-CoA之后，最終合成PHB的過(guò)程中仍有幾步反應(yīng)十分模糊，相應(yīng)的酶基因也是未知的。在紫草素生物合成中，一般認(rèn)為PHB只能來(lái)自于PP，因?yàn)槟壳霸诟叩戎参镏兴坪醪](méi)有找到與UbiC酶同源的基因與酶。此外，Sommer等[19]和Kohle等[20]將大腸桿菌的UbiC酶基因轉(zhuǎn)入了紫草毛狀根中進(jìn)行高表達(dá)，雖然紫草毛狀根中生產(chǎn)了較多的UbiC酶，但相應(yīng)的紫草素類化合物并沒(méi)顯著升高。

最新研究發(fā)現(xiàn)，紫草寧是由PP代謝途徑的產(chǎn)物PHB和MVA代謝途徑形成的GPP這兩個(gè)重要的前體，經(jīng)對(duì)羥基苯甲酸香葉基轉(zhuǎn)移酶(p-hydroxybenzoate geranyltransferase,PGT)催化形成GBA，再經(jīng)轉(zhuǎn)換為香葉基氫醌(geranyl hyrdoxyquinone, GHQ)，繼而羥基化為3?-羥基香葉基氫醌(3?-hydroxy-geranyl hyr-doxyquinone, GHQ-3?-OH)等一系列酶促反應(yīng)，最后在內(nèi)質(zhì)網(wǎng)中形成，并通過(guò)胞外分泌作用將形成的紫草寧微粒運(yùn)輸?shù)皆|(zhì)體外的細(xì)胞壁中[15,21,22]。此過(guò)程中所涉及的部分酶促反應(yīng)目前尚不清楚，亟需探究。Wang等[23,24]通過(guò)紫草細(xì)胞的轉(zhuǎn)錄組分析與生化檢測(cè)，發(fā)現(xiàn)CYP76B74催化紫草素生物合成中的香葉基氫醌3?的羥基化，為進(jìn)一步探索開(kāi)環(huán)反應(yīng)、萘醌骨架的生成鑒定了一個(gè)關(guān)鍵位置羥化酶。同時(shí)，Song等[25]通過(guò)轉(zhuǎn)錄組分析與GHQ為底物的酶催化反應(yīng)，證實(shí)CYP76B100在C-3?位置催化GHQ的香葉基側(cè)鏈羥基化，形成GHQ-3?-OH，CYP76B101在C-3?位置進(jìn)行GHQ的氧化反應(yīng)產(chǎn)生GHQ的3?-羧酸衍生物(GHQ-3?-COOH)以及GHQ-3?-OH。此外，在以紫草寧為前體的紫草寧衍生物合成通路研究中，Oshikiri等[26]通過(guò)蛋白質(zhì)組學(xué)分析鑒定到兩個(gè)關(guān)鍵的對(duì)映體特異性酰基轉(zhuǎn)移酶LeSAT1和LeAAT1，其能分別將乙?；?CoA、異丁?；?CoA和異戊酰基- CoA識(shí)別為?；w，產(chǎn)生其相應(yīng)的紫草寧/鏈烷烴衍生物即乙酰紫草寧/阿卡寧、異丁酰紫草寧/阿卡寧和異戊酰紫草寧/阿卡寧。這些研究都為進(jìn)一步探究紫草素的生物合成提供了有效信息。

圖2 紫草素的生物合成途徑

根據(jù)參考文獻(xiàn)[21]修改繪制。 ACTH: acetoacetyl-CoA thiolase，乙酰乙酰輔酶A硫解酶；C4H: cinnamic acid 4-hydroxylase，肉桂酸-4-羥化酶；4-CL: 4-coumarate:CoA ligase，4-香豆酸輔酶A連接酶；CDPMEK: 4-(cytidine 59-diphospho)-2-C-methyl-D-erythritol 2-phosphokinase，4-二磷酸胞苷-2-C-甲基-D-赤蘚醇激酶；DXPS: 1-deoxy-D-xylulose-5-phosphate synthase，1-脫氧-D-木酮糖-5-磷酸合酶；DXR: 5-phosphoric acid-deoxyxylulose reductase，5-磷酸–去氧木酮糖還原酶；GDPS: geranyldiphosphate synthase，焦磷酸香葉酯合酶；GHQH: geranylhydroquinone 3’-hydroxylase，香葉基氫醌3?-羥化酶；HDR: 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase，1-羥基-2-甲基-2-(E)-丁烯基-4-焦磷酸還原酶；HDS: 1-hydroxy-2-methyl-2-(E)-butenyl-diphosphate synthase，1-羥基-2-甲基-2-(E)-丁烯基-4-二磷酸合酶；HMGR: hydroxymethylglutaryl-coenzyme A reductase，甲戊二羥酸單酰輔酶A還原酶；HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase，3-羥基-3-甲基–戊二酰輔酶A合成酶；MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase，2-C-甲基-D-赤蘚糖醇4-磷酸胞苷轉(zhuǎn)移酶；MECDPS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, 2-C-甲基-D-赤蘚糖醇2,4-環(huán)焦磷酸合成酶；MVAK: mevalonate 5-phospho kinase，甲羥戊酸5-磷酸激酶；MVDD: mevalonate diphosphate decarboxylase，甲羥戊酸5-焦磷酸脫羧酶；PAL: phenylalanine ammonia-lyase，苯丙氨酸解氨酶；PGT: p-hydroxybenzoate geranyltransferase，對(duì)羥基苯甲酸香葉基轉(zhuǎn)移酶；PMVK: 5-phosphomevalonate phosphokinase，磷酸甲羥戊二酸激酶；SAT: shikonin acetyltransferase，紫草寧乙酰轉(zhuǎn)移酶。

3 調(diào)控紫草素生物合成的功能相關(guān)基因

先前的研究者通過(guò)抑制差減雜交(suppression subtractive hybridization, SSH)及cDNA末端快速擴(kuò)增(rapid-amplification of cDNA ends, RACE)等技術(shù)，已經(jīng)成功篩選和克隆了一系列紫草、軟紫草以及滇紫草中與紫草素生物合成直接或間接相關(guān)的酶基因和調(diào)控基因[16]。

3.1 紫草素生物合成直接相關(guān)的酶基因

由于紫草素生物合成核心部分不明確，目前已經(jīng)鑒定和克隆的與紫草素合成直接相關(guān)酶基因僅在上游部分，主要包括MVA途徑和PP途徑的一些酶基因以及催化GBA合成的基因。

在MVA途徑中，鑒定與克隆的軟紫草基因較為完整。其中軟紫草的乙酰乙酰輔酶A硫解酶基因(acetoacetyl-coenzyme A thiolase gene,)、甲戊二羥酸單酰輔酶A合成酶基因(hydroxy-methylglutaryl CoA synthase gene,)、甲戊二羥酸單酰輔酶A還原酶基因(hydroxymethylglutaryl-coenzyme A redu-ctase gene,)、甲羥戊酸焦磷酸激酶基因(phosphomevalonate kinase gene,)、甲羥戊酸激酶基因(mevalonate kinase gene,)、甲羥戊酸5-焦磷酸脫羧酶基因(mevalonate disphosphate decarboxy-lase gene,)、異戊烯焦磷酸異構(gòu)酶基因(isopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomerase gene,)以及焦磷酸香葉酯合酶基因(geranyl diphosphate syn-thase gene,)均已成功獲得[16]。但是，到目前為止，在紫草中僅鑒定和克隆了紫草甲戊二羥酸單酰輔酶A還原酶基因(hydroxyme-thylglutaryl-coenzyme A reductase gene,)[27]。而MVA途徑中，HMGR酶可能是唯一的限速酶，直接影響到紫草素的合成[15,16]。

在PP途徑中，目前已知的酶基因在紫草和軟紫草中均已經(jīng)被鑒定和克隆，即苯丙氨酸解氨酶基因(phenylalanine ammonia-lyase gene,)、肉桂酸-4-羥化酶基因(cinnamic acid 4-hydroxylase gene,)和基因[28~30]。其中，酶基因被認(rèn)是控制PP途徑的關(guān)鍵起始酶基因[30]；而酶基因與酶基因可能不直接影響紫草素的合成[28,30]。

作為紫草素合成核心部分的關(guān)鍵起始酶基因，具有非常重要的作用，其在紫草與軟紫草中均已經(jīng)被鑒定與克隆。有研究報(bào)道，許多調(diào)控因素，如光信號(hào)、植物激素(如二氯苯氧乙酸(dichlorphe-noxyacetic acid, 2, 4-D)與茉莉酸甲酯(methyl jasmonate, MJ))以及無(wú)機(jī)離子(如NH4+)，均直接作用于，從而調(diào)控紫草素的生物合成[31]。

3.2 紫草素生物合成間接相關(guān)的調(diào)控基因

除了與紫草素合成直接相關(guān)的酶基因以外，研究者還在新疆紫草和紫草中克隆了許多間接相關(guān)的調(diào)控基因，其中主要包括一些調(diào)控因素的潛在受體基因或轉(zhuǎn)錄因子基因，以及這些調(diào)控因素合成相關(guān)的酶基因。

Yazaki等[32]通過(guò)SSH技術(shù)鑒定與克隆了一系列紫草黑暗誘導(dǎo)基因(dark-inducible gene,)。其中，只有基因受到光信號(hào)的嚴(yán)格調(diào)控，在黑暗中特異表達(dá)?；虍a(chǎn)物與擬南芥的脂質(zhì)轉(zhuǎn)移蛋白(lipid-transfer protein, EARLI 1)同源，可能具有穩(wěn)定紫草素合成場(chǎng)所(即細(xì)胞內(nèi)小囊泡)的功能，但具體功能目前尚不明確。

花卉分生組織/乙烯響應(yīng)轉(zhuǎn)錄因子(APETALA2/ ethylene-responsive factor,AP2/ERF)基因家族與乙烯不敏感蛋白3/類乙烯不敏感蛋白(/protein, EIN3/EIL)基因家族是乙烯信號(hào)傳導(dǎo)途徑中關(guān)鍵的轉(zhuǎn)錄因子[33,34]。研究發(fā)現(xiàn)，紫草基因可能參與光信號(hào)和乙烯對(duì)紫草素合成的調(diào)控[35]。Fang等[36]將基因在紫草毛狀根中高表達(dá)，構(gòu)建出了一個(gè)高產(chǎn)紫草素的毛狀根體系。

Fe+結(jié)合的順式還原酮加雙氧酶基因(aciredu-ctone dioxygenase gene,)被證明參與到乙烯與多胺的合成途徑中[37]。Qi等[38]發(fā)現(xiàn)紫草基因可能參與調(diào)控紫草素的生物合成。此外，有研究報(bào)道，多胺合成途徑中的精氨酸脫羧酶基因(arginine decarboxylase gene,)也可能參與紫草素合成的調(diào)控[35]。

Yazaki等[39]鑒定了一個(gè)與病程相關(guān)蛋白(pathogenesis related protein, PR)同源的基因，其可能和紫草素合成有關(guān)。Yamamura等[40]在紫草細(xì)胞系中鑒定得到一個(gè)質(zhì)外體(細(xì)胞壁)表達(dá)的色素愈傷組織特異性基因(pigment callus-specificgene,)，并推測(cè)其可能與紫草素的分泌相關(guān)。另外，研究者還鑒定、克隆了部分紫草中MYB和MYC轉(zhuǎn)錄因子基因，認(rèn)為它們的功能可能也與紫草素合成有關(guān)[18,41,42]。

4 紫草素的生物活性及其藥理功能

根據(jù)國(guó)內(nèi)外研究報(bào)道，紫草素及其衍生物具有顯著的抗腫瘤、抗炎、抗菌、抗病毒、抗氧化等多重藥理作用，因而具有廣闊的開(kāi)發(fā)應(yīng)用前景[43~45]。

4.1 抗腫瘤活性作用

大量研究表明，紫草素及其衍生物(圖3)對(duì)不同的腫瘤細(xì)胞表現(xiàn)出顯著的細(xì)胞毒性，其抗癌作用牽涉到多個(gè)靶點(diǎn)，抗癌機(jī)制包括促細(xì)胞凋亡、誘導(dǎo)細(xì)胞壞死、抑制DNA拓?fù)洚悩?gòu)酶活性、抑制酪氨酸激酶磷酸化、抑制血管再生及調(diào)控多條與腫瘤相關(guān)的信號(hào)通路等。

SH-7是在紫草寧的結(jié)構(gòu)基礎(chǔ)上修飾的來(lái)的一種新的萘醌類化合物，它通過(guò)抑制Topo II的活性達(dá)到抗腫瘤效果，且抑制效果遠(yuǎn)比其母體化合物紫草寧好[46]。SH-7有效穩(wěn)定Topo II-DNA復(fù)合物并提高了磷酸化的組蛋白H2AX的表達(dá)量，同時(shí)，它對(duì)Topo I也有抑制作用，但效果不如Topo II。早在1995年，Ahn等[47]就合成了一系列乙酰紫草寧類似物，并發(fā)現(xiàn)這些類似物是DNA Topo I的良好抑制劑。Qiu等[48]合成的紫草素衍生物PMMB172對(duì)三陰性乳腺癌細(xì)胞MDA-MB-231的增殖有良好的抑制作用，它通過(guò)靶向信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子(signal transduc-tion and transcriptional activators, STAT3)的SH2結(jié)構(gòu)域來(lái)抑制STAT3的入核以及在細(xì)胞核中的定位，進(jìn)而抑制其下游基因的表達(dá)。

對(duì)紫草寧及其衍生物的抗腫瘤機(jī)制研究最多的要數(shù)微管蛋白。2011年，Acharya等[49]報(bào)道了萘醌能夠使微管蛋白解聚，造成紡錘體微管組織混亂，導(dǎo)致大部分細(xì)胞都被阻滯在G2/M期?？紤]到紫草寧也屬于萘醌類化合物，關(guān)于它是否為微管蛋白抑制劑的研究便成為熱點(diǎn)。2014年，Wang等[50]合成了一系列雜環(huán)羧酸紫草寧酯，并從中篩選獲得3-吲哚丙酸紫草寧酯(compound 3)和3-噻吩乙酸紫草寧酯(compound 8)兩種對(duì)HeLa細(xì)胞增殖抑制效果明顯的化合物，并推測(cè)其為良好的微管蛋白抑制劑。Guo等[51]在紫草寧支鏈羥基上引入了苯氧苯乙酸，并證明它(compound 16)是通過(guò)抑制微管蛋白的聚合來(lái)有效抑制HepG2細(xì)胞增殖的。Baloch等[52]也對(duì)紫草寧進(jìn)行結(jié)構(gòu)修飾，并篩選出化合物3j，認(rèn)為它能夠干擾微管蛋白的聚集使得HepG2細(xì)胞周期抑制在G2/M期，同時(shí)激活caspase導(dǎo)致細(xì)胞凋亡。Lin等[53]通過(guò)硫原子作為橋，在紫草寧支鏈和萘醌環(huán)上連接巰基糖，并通過(guò)實(shí)驗(yàn)證明連接兩個(gè)木糖的紫草寧巰基糖衍生物IIb的抗癌活性最佳，而且它也是通過(guò)靶向微管蛋白而發(fā)揮作用的。Sun等[54]合成獲得的苯甲酰丙烯酸紫草寧酯PMMB317可通過(guò)靶向表皮生長(zhǎng)因子受體(epidermal growth factor receptor, EGFR)和微管蛋白，發(fā)揮雙重抗腫瘤作用。

圖3 具有抗癌活性的代表性紫草寧衍生物

SH7, Compound 3, Compound 8, Compound 16, 3j, PMMB172, PMMB317, Naphthazarin, IIb均為化合物在文獻(xiàn)中的名稱及編號(hào)。

相比之下，關(guān)于阿卡寧及其衍生物的抗腫瘤活性研究相對(duì)較少(圖4)。2004年，Huang等[55]利用多種親核物質(zhì)對(duì)β, β-二甲基丙烯酰阿卡寧進(jìn)行還原烷基化和氧化共軛加成反應(yīng)，所得產(chǎn)物8、11b、12b、14b相比于母體化合物β, β-二甲基丙烯酰阿卡寧(1)和乙酰阿卡寧(2)，對(duì)人肺腺癌細(xì)胞株GLC-82、人鼻咽癌細(xì)胞株CNE2、人肝癌細(xì)胞株Bel-7402和人白血病細(xì)胞株K562具有更高的體外細(xì)胞毒性。另外，作者推測(cè)，細(xì)胞組分(親核試劑)與醌(親電試劑)的共軛加成和還原烷基化可能正是醌類結(jié)構(gòu)具有細(xì)胞毒性的原因。2010年，Deng等[56]報(bào)道一個(gè)新型的阿卡寧衍生物SYUNZ-16，稱其可通過(guò)抑制蛋白激酶B(Protein kinase B, PKB/AKT)激酶活性、阻斷蛋白激酶B/叉頭狀轉(zhuǎn)錄因子O (Protein kinase B/forkhead box O, AKT/FOXO)信號(hào)通路來(lái)誘導(dǎo)細(xì)胞凋亡和抑制腫瘤的生長(zhǎng)。Zhang等[57~59]報(bào)道了二甲基化阿卡寧衍生物S-2a、阿卡寧肟衍生物S-11、DMAKO-05等小分子具有很好的體外抗腫瘤活性。此外，Chang等[60]發(fā)現(xiàn)阿卡寧可誘導(dǎo)活性氧(reactive oxygen species, ROS)水平升高從而對(duì)DNA造成氧化損傷，同時(shí)聯(lián)合聚腺苷二磷酸–核糖聚合酶(poly- ADP-ribose polymerase, PARP)抑制劑奧拉帕尼在無(wú)毒劑量下，顯著抑制體內(nèi)外結(jié)腸癌的生長(zhǎng)。

圖4 具有抗癌活性的代表性阿卡寧衍生物

1, 2, 8, 14b, 11b, 12b, SYUN-16, S-2a, S-11, DMAKO-05均為化合物在文獻(xiàn)中的名稱及編號(hào)。

4.2 抗炎活性功能

Yang等[61]通過(guò)研究紫草寧的抗炎機(jī)制，發(fā)現(xiàn)紫草寧通過(guò)干擾素和核因子κB (nuclear factor kappa-B, NF-κB)信號(hào)通路，抑制RAW264.7細(xì)胞中脂多糖(lipopolysaccharid, LPS)誘導(dǎo)的高遷移率族蛋白B1 (high mobility group box 1, HMGB1)的釋放。此外，紫草寧和β-羥基異戊?；喜輰?β-valeryl,β-HIVS) (圖5)，通過(guò)抑制血管內(nèi)皮生長(zhǎng)因子受體(vascular endothelial growth factor receptor, VEGFR)與三磷酸腺苷(triphosadenine, ATP)非競(jìng)爭(zhēng)性的方式抑制血管生成[62]。萘醌化合物CMEP-NQ可以在不影響細(xì)胞活力的前提下，下調(diào)LPS誘導(dǎo)的誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase, iNOS)和環(huán)氧合酶-2(cyclooxygenase-2, COX-2)表達(dá)，從而抑制一氧化氮(nitric oxide, NO)和前列腺素E2 (prostaglandin E2, PGE2)的產(chǎn)生[63]達(dá)到抗炎作用。乙酰紫草寧具有抗過(guò)敏和抗炎作用，能有效降低過(guò)敏性鼻炎小鼠模型的過(guò)敏性炎癥，主要是通過(guò)抑制輔助型T細(xì)胞2 (T helper 2 cell, Th2)相關(guān)卵清蛋白(ovalbumin, OVA)特異性免疫球蛋白E (immune globulin, IgE)、IgG1的產(chǎn)生，同時(shí)抑制Th2細(xì)胞產(chǎn)生白細(xì)胞介素4 (interleukin-4, IL-4)、IL-5、IL-13和肥大細(xì)胞產(chǎn)生組胺；此外，乙酰紫草寧可減輕炎癥細(xì)胞浸潤(rùn)和杯狀細(xì)胞增生程度[64]。自噬在乙酰紫草寧(圖5)通過(guò)腺苷酸活化蛋白激酶/哺乳動(dòng)物雷帕霉素靶蛋白(adenosine monophosphate activated protein kinase/mammalian target of rapamycin, AMPK/Mtor)通路對(duì)非酒精性脂肪性肝炎的治療起著關(guān)鍵作用，Zeng等[65]發(fā)現(xiàn)乙酰紫草寧不僅可以改善脂肪變性，還可能通過(guò)誘導(dǎo)飲食缺乏蛋氨酸膽堿的小鼠肝臟自噬來(lái)減輕肝臟炎癥、肝損傷和肝纖維化。Cui等[66]研究證明乙酰紫草寧可明顯減弱動(dòng)脈粥樣硬化模型小鼠斑塊內(nèi)炎性細(xì)胞(T淋巴細(xì)胞、中性粒細(xì)胞、巨噬細(xì)胞)浸潤(rùn)，IL-1、IL-6、腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α)和單核細(xì)胞趨化蛋白-1 (monocyte chemotactic protein 1, MCP-1)水平降低，并通過(guò)抑制NF-κB信號(hào)通路改善小鼠血管炎癥。Zhang等[67]使用紫草羥基萘醌類混合物(hydroxy-naphthoquinone mixture, HM)治療葡聚糖硫酸鈉(dextran sulfate sodium, DSS)誘導(dǎo)的潰瘍性結(jié)腸炎，結(jié)果顯示，HM能有效改善小鼠結(jié)腸炎臨床癥狀和組織病理?yè)p傷。早在2012年，西班牙學(xué)者Andújar等[68]首次報(bào)道了紫草寧對(duì)DSS誘導(dǎo)的實(shí)驗(yàn)性結(jié)腸炎具有顯著治療作用，并證明紫草寧是通過(guò)阻斷NF-κB和STAT3信號(hào)表達(dá)，以及它們的促炎反應(yīng)，來(lái)調(diào)節(jié)結(jié)腸炎和與之相關(guān)的腫瘤發(fā)生。

4.3 抗菌作用研究

最初，人們發(fā)現(xiàn)紫草根部的粗提物具有抗菌活性，在過(guò)去五十年內(nèi)，很多研究人員對(duì)紫草根部活性成分的抗菌活性做了更深入、系統(tǒng)的研究，并得出結(jié)論紫草寧及其衍生物具有廣譜的抗菌活性。目前，紫草寧及其衍生物已被證明對(duì)革蘭氏陽(yáng)性菌有明顯的抗菌活性，如金黃色葡萄球菌、腸球菌、枯草芽孢桿菌等，最小抑菌濃度(minimum inhibitory concentration, MIC)值大約在0.30~6.25 μg/mL之間。相反，它們對(duì)革蘭氏陰性菌，如大腸桿菌、綠膿假單胞菌和黃色微球菌等沒(méi)有明顯的作用[69]。

早期文獻(xiàn)報(bào)道紫草寧及其衍生物具有抑菌作用，但是近期的動(dòng)力學(xué)研究說(shuō)明紫草寧及其衍生物是具有殺菌作用的[70]。Shen等[71]的研究表明紫草寧(圖5)可用于治療對(duì)甲氧西林有抗性的金黃色葡萄球菌，MIC值為6.25 μg/mL。后期的研究發(fā)現(xiàn)紫草寧酯類衍生物對(duì)金黃色葡萄球菌有更好的活性，如甲基丁酰紫草寧(圖5)，其對(duì)金黃色葡萄球菌的MIC值為1.56 μg/mL。Kuo等[72]還報(bào)道了紫草寧以濃度依賴的方式抑制幽門螺旋桿菌的增殖。一些腸內(nèi)細(xì)菌包括肺炎桿菌、沙門氏菌、金黃色葡萄球菌和大腸桿菌等體內(nèi)的酶會(huì)表現(xiàn)N-乙?；D(zhuǎn)移酶(N-acetyltrans-ferase, NAT)活性，有助于化學(xué)致癌物質(zhì)的代謝激活，Kuo等[72]研究人員還發(fā)現(xiàn)了紫草寧可以抑制NAT-介導(dǎo)的N-乙?；瑥亩钄郚AT對(duì)化學(xué)致癌物質(zhì)的激活。

4.4 抗病毒活性和其它藥理功能

紫草寧及其衍生物抗病毒研究也由來(lái)已久。2003年，Chen等[73]的研究顯示紫草寧可下調(diào)巨噬細(xì)胞表面一個(gè)HIV-1的主要共同受體趨化因子受體5 (chemokine receptor, CCR5)的表達(dá)從而抑制HIV病毒的復(fù)制。2017年，Zhang等[74,75]發(fā)現(xiàn)紫草寧衍生物PMM034可顯著抑制橫紋肌肉瘤(rhabdomyosar-coma, RD)細(xì)胞中促炎因子的表達(dá)水平，從而抑制引起手足口病的71型人腸病毒(human enterovirus 71 of hand, foot and mouth disease, EV71)的活性。此外，他們還發(fā)現(xiàn)PMM034可通過(guò)抑制神經(jīng)氨酸苷酶有效抑制流感病毒H1N1的表達(dá)，且效果可媲美于陽(yáng)性藥奧司他韋[74,75]。

圖5 具有抗炎、抗菌活性的代表性紫草寧及其衍生物

Shikonin (紫草寧)，Acetylshikonin (乙酰紫草寧)，2-Methylbutyryl shikonin (2-甲基丁酰紫草寧)，β-hydroxyisovaleryl shikonin (β-羥基異戊酰紫草寧)，CMEP-NQ (萘醌化合物)均為化合物在文獻(xiàn)中的名稱及編號(hào)。

Lee等[76,77]發(fā)現(xiàn)紫草寧可通過(guò)WNT/-catenin途徑顯著抑制3T3-L1細(xì)胞中脂肪的形成和積累，并由此推測(cè)其同樣可以利用該通路對(duì)肥胖癥及相關(guān)疾病起到治療作用。Wang等[78]研究發(fā)現(xiàn)紫草寧可通過(guò)抗氧化作用對(duì)小鼠腦出血、再灌注損傷起到保護(hù)作用。從機(jī)制上來(lái)說(shuō)，即紫草寧顯著降低了神經(jīng)系統(tǒng)缺陷評(píng)分、梗死面積、丙二醛(malondialdehyde, MDA)、羰基和活性氧的水平，減弱了神經(jīng)元損傷，上調(diào)了超氧化物歧化酶(superoxide dismutase, SOD)、過(guò)氧化氫酶(catalase, CAT)、谷胱甘肽過(guò)氧化物酶(glutathioneperoxidase, GSH- px)的活性，降低了谷胱甘肽(glutathione, GSH)/谷胱甘肽二硫化合物(glutathione disulfide, GSSG)的比例。

5 結(jié)語(yǔ)與展望

紫草作為我國(guó)傳統(tǒng)中藥，國(guó)內(nèi)外對(duì)其次生代謝產(chǎn)物—紫草素的生物合成與分子代謝研究日益精進(jìn)。紫草素除了在藥理作用方面顯示良好的活性外，同時(shí)也是名貴的染料，作為天然食用色素或添加到化妝品中加以使用，基于此，當(dāng)前面臨著臨床或生產(chǎn)需求量大與珍貴植物物種資源日益匱乏的矛盾。因此，亟需從不同角度入手提高紫草寧及其衍生物的產(chǎn)量，實(shí)現(xiàn)規(guī)模化生產(chǎn)紫草素：一方面，雖然紫草寧及其衍生物的合成途徑已基本清楚，但涉及到控制催化GHQ-3?-OH合成紫草寧過(guò)程以及紫草寧合成其一系列衍生物的關(guān)鍵酶基因尚不明確，已知的合成途徑中關(guān)鍵基因的表達(dá)調(diào)控也有待探明，因此，有必要通過(guò)基因組學(xué)、轉(zhuǎn)錄組學(xué)、蛋白質(zhì)組學(xué)和代謝組學(xué)等多組學(xué)研究相結(jié)合，明晰紫草寧及其衍生物合成調(diào)控通路中尚未發(fā)現(xiàn)的關(guān)鍵酶基因，繼而通過(guò)基因工程或代謝工程手段，獲得可高效生產(chǎn)紫草寧及其衍生物的高產(chǎn)轉(zhuǎn)基因株系，用于生產(chǎn)實(shí)踐；另一方面，可在明晰紫草寧及其衍生物生物合成調(diào)控途徑的基礎(chǔ)上，采用合成生物學(xué)的方法，通過(guò)改造工程菌株使其合成分泌紫草寧及衍生物；或者，還可通過(guò)增加高產(chǎn)紫草素等藥用成分的紫草科藥用紫草的廣泛栽培，用以緩解野生植物種質(zhì)資源與紫草素的供求矛盾。同時(shí)，國(guó)內(nèi)外學(xué)者針對(duì)紫草素進(jìn)行的抗腫瘤、抗炎、抗菌、抗病毒等藥理研究，逐步挖掘了其潛在的分子作用機(jī)制。然而到目前為止，仍然沒(méi)有找到明確的十分高效的作用靶點(diǎn)，以提高紫草素類天然產(chǎn)物對(duì)腫瘤的靶向性。此外，紫草素類天然產(chǎn)物抗細(xì)菌、真菌及病毒的確切分子機(jī)制也并不十分明確，這嚴(yán)重阻礙了其藥用價(jià)值的開(kāi)發(fā)。根據(jù)前人的研究，紫草素在抗炎、抗癌方向?qū)I3K/AKT/mTOR、MAPK、JAK/STAT、NF-κB通路的影響較為顯著[79~84]；在抗菌方面紫草素對(duì)生物膜的形成和成熟抑制、誘導(dǎo)群體感應(yīng)分子法尼醇的產(chǎn)生、增加內(nèi)源性活性氧(ROS)的產(chǎn)生、阻斷組蛋白H3去乙?；?、導(dǎo)致內(nèi)源性NO積累等都是研究的熱點(diǎn)[85~88]。隨著現(xiàn)代分子生物學(xué)、合成生物學(xué)及生物信息學(xué)的快速發(fā)展，相信明確紫草素的完整生物合成途徑及其調(diào)控、挖掘紫草素的潛在作用靶點(diǎn)將不是難題；并根據(jù)可能的作用靶點(diǎn)，再進(jìn)行深入的化學(xué)結(jié)構(gòu)或生化功能等各種修飾，可望極大地促進(jìn)中藥植物天然產(chǎn)物生物合成的調(diào)控，有效地開(kāi)發(fā)紫草素或其衍生物成為臨床應(yīng)用的植物源新藥。

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Progress on biosynthesis and function of the natural products of Zi Cao as a traditional Chinese medicinal herb

Hongyan Lin, Xuan Wang, Cong He, Ziling Zhou, Minkai Yang, Zhongling Wen, Hongwei Han, Guihua Lu, Jinliang Qi, Yonghua Yang

Zi Cao is an important traditional medicinal plant resource in China. Shikonin and its derivatives, as the purple-red naphthoquinones among natural products of its roots, are commonly used clinically in the treatment of sores and skin inflammations. Over the past few decades, due to their highly effective multiple biological activities, pharmacological effects, good clinical efficacy and high utilization value, shikonin and its derivatives have attracted increasing attention of domestic and foreign researchers. For this reason, the wild plant germplasm resources have been suffering a grievous exploitation, leading to a serious threat to the habitat. With the development of the biosynthesis, molecular metabolism and biotechnology, as well as the continuous innovation of research methods on the biological activities and pharmacological effects of plant natural products, significant progress has been made in the research on the biosynthetic pathways and related regulatory genes of shikonin. The pharmacological action and its mechanism of shikonin have also been deeply elucidated, which greatly promoted the basic research and clinical application development of shikonin. In this review, we briefly introduce and analyze the classification of Zi Cao, structure and composition of natural shikonin and its biosynthesis pathway, functional genes related to the regulation of shikonin biosynthesis, and biological activities and pharmacological functions of shikonin. Finally, we address possible prospective for the trend on the future research and development of natural shikonin and its derivatives, hoping to provide a useful reference for the deep mining and development of medicinal natural products from important Chinese medicinal materials, and to promote the modern development of traditional Chinese medicine.

Zi Cao; shikonin; biosynthesis; gene regulation; pharmacological activity

2020-10-10;

2021-03-04

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：U1903201, 31670298, 31771413, 21702100, 21907051)和教育部創(chuàng)新團(tuán)隊(duì)項(xiàng)目(編號(hào)：IRT_14R27)資助[Supported by the National Natural Science Foundation of China (Nos. U1903201, 31670298, 31771413, 21702100, 21907051), and the Program for Changjiang Scholars and Innovative Research Team in University from the Ministry of Education of China (No. IRT_14R27)]

林紅燕，博士，助理研究員，研究方向：藥用植物天然產(chǎn)物化學(xué)和分子藥理。E-mail: linhy@nju.edu.cn

王煊，博士研究生，研究方向：植物分子代謝。E-mail: dg1930043@smail.nju.edu.cn

林紅燕和王煊并列第一作者。

楊永華，教授，博士生導(dǎo)師，研究方向：分子代謝與生物技術(shù)安全。E-mail: yangyh@nju.edu.cn

10.16288/j.yczz.20-341

2021/3/29 11:37:11

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20210326.0956.002.html

(責(zé)任編委: 陳德富)