張樹(shù)庭 Solomon P. Wasser

（1.國(guó)際蕈菌生物技術(shù)服務(wù)中心，香港中文大學(xué)生物系，香港；2. 國(guó)際生物多樣性與真菌生物技術(shù)中心，海法大學(xué)自然科學(xué)院進(jìn)化研究所進(jìn)化與環(huán)境生物學(xué)系，以色列，海法；烏克蘭國(guó)家科學(xué)院國(guó)家科學(xué)院霍洛德內(nèi)植物研究所，烏克蘭，基輔）

食藥用菌保健金字塔模型對(duì)增進(jìn)人類(lèi)福祉的作用（七）

張樹(shù)庭1Solomon P. Wasser2

（1.國(guó)際蕈菌生物技術(shù)服務(wù)中心，香港中文大學(xué)生物系，香港；2. 國(guó)際生物多樣性與真菌生物技術(shù)中心，海法大學(xué)自然科學(xué)院進(jìn)化研究所進(jìn)化與環(huán)境生物學(xué)系，以色列，海法；烏克蘭國(guó)家科學(xué)院國(guó)家科學(xué)院霍洛德內(nèi)植物研究所，烏克蘭，基輔）

醫(yī)藥公司的藥品研發(fā)需要新的天然產(chǎn)品來(lái)源。藥用蕈菌就是一種最好的天然公共資源，它在較短的時(shí)間內(nèi)即可收獲并用于新藥的研制。

下面詳細(xì)地闡述蕈菌藥品的發(fā)展過(guò)程。蕈菌生物活性代謝產(chǎn)物的藥品發(fā)現(xiàn)途徑包括以下基本步驟[14]。

8.1 蕈菌培養(yǎng)和生物質(zhì)生產(chǎn)

首先，經(jīng)選擇的目標(biāo)蕈菌菌株在固體培養(yǎng)基（瓊脂-麥芽培養(yǎng)基或酵母-瓊脂培養(yǎng)基）培養(yǎng)皿中作純菌絲培養(yǎng)。不同蕈菌種類(lèi)的菌絲體生長(zhǎng)所需時(shí)間不同，主要取決于物種特性和培養(yǎng)條件，如基質(zhì)營(yíng)養(yǎng)成分、pH值、溫度等。因此，根據(jù)研究的主要目的，應(yīng)選擇最佳生長(zhǎng)條件，使目標(biāo)菌株能夠獲得最高的生物量和生物活性物質(zhì)。一般來(lái)說(shuō)，在固體培養(yǎng)基上生長(zhǎng)的蕈菌，其菌絲培養(yǎng)溫度需維持在27 ℃，直至培養(yǎng)皿表面長(zhǎng)滿菌絲。當(dāng)純菌絲培養(yǎng)完成后，進(jìn)入第二階段，即獲得菌絲生物質(zhì)的液體發(fā)酵階段。將新鮮的菌絲培養(yǎng)物切成小塊接種于液體培養(yǎng)基（除不加瓊脂外，其他營(yíng)養(yǎng)成分相同）中，并置于旋轉(zhuǎn)搖床上生長(zhǎng)，直到菌絲球填滿整個(gè)培養(yǎng)基。該培養(yǎng)階段所需時(shí)間視不同種類(lèi)蕈菌而異。為擴(kuò)大生產(chǎn)量，需將得到的菌絲體勻漿接種到更大容器的液體培養(yǎng)基中，并置于搖床（或發(fā)酵罐）中培養(yǎng)。

8.2 生物提取

從蕈菌液體培養(yǎng)中獲得的生物質(zhì)需作干燥處理，并研磨成粉末。根據(jù)所提取蕈菌化合物的種類(lèi)，需選擇不同的有機(jī)溶劑。例如，要透過(guò)細(xì)胞膜萃取化合物，應(yīng)選擇不溶于水的溶劑，如乙酸乙酯。相反，若提取細(xì)胞表面化合物，則應(yīng)選擇溶于水的溶劑，如醇類(lèi)。一般來(lái)說(shuō)，最常見(jiàn)的用于生物質(zhì)提取的有機(jī)溶劑有甲醇、乙醇、乙酸乙酯、乙醚、福爾馬林等，或是其中某幾種溶劑的組合。此外，由于培養(yǎng)液中含有豐富的蕈菌次生代謝活性物質(zhì)，因此也可以用不溶于水的溶劑，如乙酸乙酯等從中萃取活性物質(zhì)。

8.3 蕈菌萃取物的篩選

上面提到的蕈菌提取物的初步篩選分離可以采用多種不同的方法和檢測(cè)分析技術(shù)。根據(jù)主要研究目標(biāo)，需篩選出特定的癌細(xì)胞系。其中一種可能的方法是利用熒光素酶活性報(bào)告基因分析。攜帶在特異性啟動(dòng)子控制下的熒光素酶活性報(bào)告基因的轉(zhuǎn)染細(xì)胞應(yīng)該加以利用。在這種情況下，可采用分光光度計(jì)法測(cè)定提取物的活性，計(jì)算報(bào)告基因抑制率的百分比。另一種檢測(cè)提取物對(duì)癌細(xì)胞存活率影響效果的方法是細(xì)胞增殖試驗(yàn)，用提取物處理細(xì)胞一段時(shí)間后，用臺(tái)盼藍(lán)染色劑排除法或MTT和XTT法測(cè)定活細(xì)胞的量。計(jì)算出代表提取物活性的癌細(xì)胞生長(zhǎng)抑制率和IC50（抑制50％癌細(xì)胞生長(zhǎng)的提取物濃度）。初步篩選試驗(yàn)至少需2次重復(fù)。

報(bào)告基因抑制率高于50％和癌細(xì)胞生長(zhǎng)率低于50％的提取物，可被看作是有效的，應(yīng)進(jìn)行第二輪篩選，以確認(rèn)其功效。第二輪篩選，可采用相同的方法，但需對(duì)初選獲得的提取物作更多的濃度試驗(yàn)，以便更加精確地評(píng)估其效果。

8.4 選定提取物對(duì)靶標(biāo)的影響

檢測(cè)并記錄與癌細(xì)胞的生長(zhǎng)、增殖、轉(zhuǎn)移、耐藥性等相關(guān)的靶細(xì)胞蛋白水平，反映選定提取物的效果。接種癌細(xì)胞，用一定濃度的提取物對(duì)其進(jìn)行處理、收集和裂解。采用蛋白質(zhì)印跡（western blot）和酶聯(lián)免疫吸附測(cè)定（ELISA）方法可檢測(cè)出目標(biāo)蛋白。特定基因的表達(dá)涉及腫瘤發(fā)生過(guò)程，也可用聚合酶鏈反應(yīng)（PCR）檢測(cè)靶基因的表達(dá)，用RT-PCR技術(shù)建立基因表達(dá)的大致水平，再用實(shí)時(shí)定量PCR技術(shù)測(cè)定該基因表達(dá)的準(zhǔn)確水平。目標(biāo)蛋白水平和靶基因水平的確立與蕈菌提取物的作用效果一致，可說(shuō)明細(xì)胞內(nèi)水平的特定作用機(jī)理。然后，選擇對(duì)靶標(biāo)有顯著作用的、最有效的提取物作進(jìn)一步的評(píng)估，并進(jìn)行化學(xué)分離，以測(cè)定有效基團(tuán)的化學(xué)起源。

8.5 選定提取物的化學(xué)分離

通過(guò)一個(gè)可使每種化合物分流成不同相態(tài)的系統(tǒng)將最有效的提取物分離為不同的化合物組分，能完成這一程序的系統(tǒng)是色譜儀。其中的阻滯系統(tǒng)可以是表面吸附劑，如二氧化硅、氧化鋁、纖維素或木炭等能夠可逆吸附的化合物?？捎酶咝б合嗌V法（HPLC），即一種分離蛋白質(zhì)和其他分子的技術(shù)，或薄層色譜法（TLC）和離子交換色譜法進(jìn)行化學(xué)分餾。這樣可獲得確切的化學(xué)組分含量，如蛋白質(zhì)、多糖、脂類(lèi)等。

8.6 確證活性餾分（化合物）的作用機(jī)理和效果

對(duì)提取物化學(xué)分餾后獲得的所有餾分進(jìn)行靶標(biāo)作用效果的評(píng)估，以篩選出含有最有效物質(zhì)的餾分。評(píng)估方法可采用蛋白質(zhì)印跡、ELISA、細(xì)胞增殖分析、PCR、RT-PCR、real-time PCR等技術(shù)。再次測(cè)定對(duì)目標(biāo)蛋白質(zhì)有積極影響的有效蕈菌餾分，以確定其特定的作用機(jī)理。

8.7 動(dòng)物試驗(yàn)

對(duì)那些在體外試驗(yàn)中具有預(yù)期效果的餾分作進(jìn)一步的動(dòng)物試驗(yàn)，以檢測(cè)其活體試驗(yàn)效果。常用于試驗(yàn)的有大鼠和裸小鼠（即用于接受人類(lèi)或其他動(dòng)物腫瘤或移植細(xì)胞的小鼠）。腫瘤細(xì)胞將被皮下注射入實(shí)驗(yàn)動(dòng)物體內(nèi)并培養(yǎng)一段時(shí)間。有效蕈菌餾分（化合物）將被注入該腫瘤動(dòng)物體內(nèi)，定期檢測(cè)其腫瘤細(xì)胞生長(zhǎng)和轉(zhuǎn)移的情況。當(dāng)該動(dòng)物死亡后，將腫瘤取出，用含有蕈菌化合物的培養(yǎng)液處理，以測(cè)定其對(duì)腫瘤細(xì)胞的直接效應(yīng)。

對(duì)在動(dòng)物活體試驗(yàn)中顯示出顯著抗癌活性的餾分進(jìn)行進(jìn)一步化學(xué)分析，以確定目標(biāo)生物活性化合物的精確化學(xué)結(jié)構(gòu)。利用藥物化學(xué)（分子的化學(xué)修飾）方法，以獲得更好和更有效的化合物。這一化合物將成為一種潛在的候選藥物。

8.8 臨床前藥物開(kāi)發(fā)

候選藥物經(jīng)過(guò)配方預(yù)制及制定，以及代謝、毒性、生物藥效率和其他藥理學(xué)性質(zhì)的測(cè)試，傳統(tǒng)上這些工作多采用動(dòng)物試驗(yàn)，而今越來(lái)越多的是在離體模型試驗(yàn)中完成。如果可以獲得預(yù)期效果，并通過(guò)產(chǎn)品穩(wěn)定性測(cè)定，那么就可以將該候選藥物用于試制生產(chǎn)。

8.9 臨床藥物開(kāi)發(fā)

最終的藥物開(kāi)發(fā)包括以下幾個(gè)階段。首先，制造的候選藥物需要通過(guò)申請(qǐng)臨床試驗(yàn)的批準(zhǔn)。之后的臨床試驗(yàn)分為三個(gè)階段：（1）在志愿者中測(cè)試藥物的安全性；（2）找出對(duì)患者有療效的佐證，估算適合的劑量；（3）準(zhǔn)確評(píng)估候選藥物的臨床應(yīng)用價(jià)值。這些階段包含了系統(tǒng)而大量的試驗(yàn)記錄，用以獲得候選藥物的市場(chǎng)準(zhǔn)入許可[14]。

9 結(jié)束語(yǔ)

食用和藥用蕈菌作為潛在的人類(lèi)食品和醫(yī)藥產(chǎn)品開(kāi)發(fā)的重要來(lái)源，前景廣闊。因此，人類(lèi)應(yīng)盡一切努力最大限度地利用農(nóng)業(yè)廢棄物資源來(lái)生產(chǎn)蕈菌。當(dāng)前我們應(yīng)齊心協(xié)力地致力于蕈菌栽培技術(shù)的研究和開(kāi)發(fā)。同時(shí)，我們應(yīng)給各國(guó)政府和國(guó)際組織，以及各產(chǎn)業(yè)界一個(gè)提醒：在蕈菌產(chǎn)業(yè)基礎(chǔ)之上開(kāi)展的研究和開(kāi)發(fā)不是一項(xiàng)奢侈的投資，而是一個(gè)國(guó)家的必備產(chǎn)業(yè)。盡管在蕈菌種植中還存在許多問(wèn)題，但是我們可以預(yù)見(jiàn)，蕈菌的利用具有更加重要和積極的效應(yīng)，尤其是作為一種穩(wěn)定的蛋白質(zhì)來(lái)源，既可以豐富人類(lèi)的飲食，又可用作膳食補(bǔ)充劑，提高人體免疫功能。這對(duì)欠發(fā)達(dá)國(guó)家尤為重要，因?yàn)榕c大多數(shù)發(fā)達(dá)國(guó)家相比，這些國(guó)家的蛋白質(zhì)和醫(yī)療保健機(jī)制較為缺乏。毫無(wú)疑問(wèn)，無(wú)論是城市還是農(nóng)村，都可以從這項(xiàng)技術(shù)中獲利，能夠以最小的成本、最大限度地提高單位面積的蕈菌產(chǎn)量，從而利用農(nóng)業(yè)廢棄物獲得一個(gè)新的生產(chǎn)蛋白質(zhì)食品的廉價(jià)來(lái)源；同時(shí)開(kāi)啟了蕈菌生物技術(shù)加工的旅程，為人類(lèi)提供可促進(jìn)健康的膳食補(bǔ)充劑產(chǎn)品。

過(guò)去20年的研究和生產(chǎn)利用的進(jìn)展表明，蕈菌對(duì)人類(lèi)食物和醫(yī)療保健體系都具有重要的作用?，F(xiàn)代醫(yī)學(xué)可能也有無(wú)法解決的問(wèn)題，蕈菌膳食補(bǔ)充劑有望成為一種有效緩解和預(yù)防疾病的產(chǎn)品。疾病的預(yù)防很重要，不僅可提高社會(huì)和經(jīng)濟(jì)效益，而且有利于保持/提高生命質(zhì)量和人類(lèi)的尊嚴(yán)。希望我們微弱的呼吁是對(duì)全球蕈菌產(chǎn)業(yè)提出的挑戰(zhàn)，并能對(duì)積極實(shí)施引起良好的關(guān)注和思考。

蕈菌衍生物產(chǎn)品既不是營(yíng)養(yǎng)保健品（食品/功能性食品），也不是藥品，這是因?yàn)槠渲写蟛糠之a(chǎn)品的有效成分不是常規(guī)藥物治療中所定義的單一的化合物。因此，它們可被列入膳食補(bǔ)充劑或介于食品和藥品之間的傳統(tǒng)藥物。

一些關(guān)于藥用蕈菌衍生物研究的報(bào)告稱(chēng)，藥用蕈菌產(chǎn)品如靈芝、冬蟲(chóng)夏草等，對(duì)所有的疾病有療效。我們認(rèn)為這有些言過(guò)其實(shí)。實(shí)際上，無(wú)論是常規(guī)的還是傳統(tǒng)的醫(yī)學(xué)，沒(méi)有一種已知的治療方式可以在任何人和任何疾病上通用。

我們不應(yīng)該假設(shè)蕈菌衍生物產(chǎn)品可以替代現(xiàn)代醫(yī)學(xué)。但是我們可以肯定，通常蕈菌衍生物是一種特殊類(lèi)型，既可以提高患者的總體幸福感，也可以配合現(xiàn)代醫(yī)學(xué)，起到輔助治療的作用。

如前所述，蕈菌被用作保健食品、滋補(bǔ)品和藥品（藥物和植物性藥物）。定期攝入蕈菌產(chǎn)品和蕈菌衍生物產(chǎn)品，可以使身體更健康、舒適和快樂(lè)。

蕈菌中含有豐富的藥用成分，尤其是硒（抗氧化劑）的含量高，硒可以使人更顯年輕和長(zhǎng)壽。本文主要傳遞蕈菌生產(chǎn)和使用的科學(xué)、經(jīng)濟(jì)、健康和環(huán)保等價(jià)值。

我們已經(jīng)討論了在世界的不同國(guó)家和地區(qū)，包括美國(guó)、歐盟、加拿大、澳大利亞、新西蘭、日本和中國(guó)大陸等，對(duì)引進(jìn)和管理從藥用蕈菌中獲得的膳食補(bǔ)充劑所涉及的立法和監(jiān)管問(wèn)題，以及世界衛(wèi)生組織的指導(dǎo)方針。大部分關(guān)注的還是烹飪用的藥用蕈菌的安全性和多樣性。當(dāng)前，市場(chǎng)上藥用蕈菌膳食補(bǔ)充劑產(chǎn)品的類(lèi)型非常多樣，但是用以保證其質(zhì)量的標(biāo)準(zhǔn)規(guī)范卻很少。必須進(jìn)行透徹分析研究、提高品質(zhì)、規(guī)范管理，才能增加和維持消費(fèi)者的信任，促使國(guó)家相關(guān)管理部門(mén)建立對(duì)當(dāng)前和未來(lái)蕈菌市場(chǎng)的監(jiān)管體系。我們希望現(xiàn)有的和未來(lái)的法規(guī)與標(biāo)準(zhǔn)，能夠繼續(xù)為質(zhì)量穩(wěn)定的藥用蕈菌產(chǎn)品的市場(chǎng)供應(yīng)提供保障，希望研發(fā)最好、最先進(jìn)的生物技術(shù)，以獲得穩(wěn)定、安全的蕈菌產(chǎn)品。

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