左園園 - 任佳麗 - 李忠海 -

(中南林業(yè)科技大學(xué)，湖南 長沙 410004)

食用菌是一類營養(yǎng)豐富、可供食用的大型真菌的總稱。由于獨(dú)特的口感風(fēng)味和較高的營養(yǎng)價(jià)值[1]，食用菌一直是人們?nèi)粘ｏ嬍持械闹匾M成部分。高等真菌屬于“創(chuàng)造系數(shù)”特別高的生物，含有多種結(jié)構(gòu)類型和多種生理活性的次生代謝產(chǎn)物，為人類開發(fā)新型的醫(yī)藥、農(nóng)藥、保健食品、化妝品等提供了重要的新資源[2]。真菌的子實(shí)體、菌絲體和發(fā)酵液中均富含多種活性成分，如酚酸、萜類、多糖、外源凝集素、類固醇、糖蛋白、維生素類、嘌呤類和有機(jī)酸等[3-4]。許多研究證明，真菌提取物和次生代謝產(chǎn)物具有多種顯著藥用價(jià)值如抗氧化[5-6]、抗腫瘤[7-8]、抗菌[9]、免疫調(diào)節(jié)[10]和抗炎[8,11-14]等。

中國真菌資源豐富，2013年食用菌產(chǎn)量達(dá)3.169 7×107t，占全球總產(chǎn)量70%以上[15]。充分利用真菌資源，從中篩選抗炎活性物質(zhì)具有很大潛力。本文擬對真菌中篩選出的具有抗炎作用的甾醇類物質(zhì)的來源、提取分離方法及其抗炎作用機(jī)理進(jìn)行整理和概述，為后期抗炎成分的篩選和研究提供一些思路和參考。

1 甾醇的定義及其分類

甾醇(sterol)也叫固醇，是含有羥基的類固醇，有3個(gè)己烷環(huán)和1個(gè)環(huán)戊烷，廣泛存在于動(dòng)植物和真菌中[16]。按照原料來源可分為動(dòng)物性甾醇(膽固醇)、植物性甾醇(谷甾醇、豆甾醇、菜油甾醇)和菌類甾醇(麥角甾醇)。

自然界中的甾醇以游離態(tài)或結(jié)合態(tài)存在。麥角甾醇(5,7,22-ergostatrien-3β-ol)是高等真菌中主要的甾醇[17-18]，其環(huán)結(jié)構(gòu)具有2個(gè)雙鍵，而植物甾醇環(huán)結(jié)構(gòu)只有1個(gè)雙鍵，見圖1、2。結(jié)合態(tài)中，麥角甾醇的3β-羥基與1個(gè)脂肪酸或羥基肉桂酸發(fā)生酯化，或與1個(gè)己糖(通常是葡萄糖)或6-脂肪?；禾前l(fā)生糖化[19]。單品麥角甾醇為白色或無色的片狀或針狀晶體，在280～320 nm紫外光下能產(chǎn)生各種光解產(chǎn)品，主要是維生素D2(麥角鈣化醇)、速甾醇和光甾醇[20]，根據(jù)Woodward-Fieser規(guī)則計(jì)算，麥角甾醇的最大吸收波長為283 nm。

圖1 植物甾醇基本結(jié)構(gòu)Figure 1 The fundamental structure of phytosterol

圖2 麥角甾醇結(jié)構(gòu)Figure 2 The structure of ergosterol

2 甾醇的提取、測定和純化

2.1 甾醇的提取

回流提取是最經(jīng)典的麥角甾醇提取方法，成本低且方法成熟，但提取時(shí)間長、操作過程復(fù)雜、有機(jī)溶劑消耗量大。張萱等[21]通過乙醇回流皂化提取靈芝中的麥角甾醇并采用正交試驗(yàn)對提取工藝進(jìn)行優(yōu)化。超聲波提取法操作簡單，環(huán)保高效，而且可以避免高溫對甾醇造成影響，是目前麥角甾醇研究中非常普遍的提取方法。國家藥典[22]中豬苓的麥角甾醇提取就采用該方法。趙英永[23]通過試驗(yàn)證實(shí)傳統(tǒng)浸提法沒有超聲提取的效率高。微波輔助提取在目前天然產(chǎn)物的開發(fā)研究中也應(yīng)用十分廣泛。高虹等[24]測定姬松茸中麥角甾醇的含量使用了微波輔助提取方法，并比較了傳統(tǒng)回流和超聲波提取，結(jié)果顯示微波提取更加完全。超臨界流體CO2萃取法和加壓溶劑提取法與傳統(tǒng)提取方法相比具有很多優(yōu)點(diǎn)，兩者都屬于綠色環(huán)保的提取技術(shù)，在醫(yī)藥、食品、環(huán)境分析等方面有很大的應(yīng)用價(jià)值[25]。宋師花等[26]通過HPLC測定含量，對靈芝中麥角甾醇的超臨界CO2萃取法進(jìn)行了優(yōu)化。李鵬等[27]利用加壓溶劑提取法對天然和人工冬蟲夏草中的麥角甾醇進(jìn)行了提取。但是這2種方法成本都很高，在現(xiàn)有的技術(shù)下還不能夠大規(guī)模的應(yīng)用。

2.2 甾醇的測定

麥角甾醇溶液不穩(wěn)定，對照品和樣品溶液都應(yīng)避光低溫保存，且不應(yīng)存放太長時(shí)間，盡量現(xiàn)配現(xiàn)測[28]。菌菇中麥角甾醇的含量研究已經(jīng)有了一些成果，雞油菌、喇叭菌、美味牛肝菌、松乳菇、冬蟲夏草等已有研究[17-18,29]。甾醇含量測定的常用方法有高效液相色譜法、分光光度法、氣相色譜和薄層掃描法等。與其他方法相比，HPLC 分析不需要進(jìn)行衍生化，操作簡單，結(jié)果可靠。Villares等[17]驗(yàn)證了HPLC法測量松露中麥角甾醇含量的方法，并討論了結(jié)合態(tài)麥角甾醇的含量。李向敏[30]35-47通過高效液相測定了5種藥用真菌中麥角甾醇的含量。Barreira等[31]使用反相高效液相色譜和紫外檢測(HPLC-UV)提取和分析了多種蘑菇甾醇，并對該方法進(jìn)行了優(yōu)化和驗(yàn)證。薄層掃描法曾用于麥角甾醇的含量測定，但是該法步驟繁雜且耗時(shí)較長。Matilla等[20]通過氣相色譜分析內(nèi)標(biāo)法對栽培蘑菇和野生蘑菇麥角甾醇含量進(jìn)行了比較。

2.3 甾醇的分離純化

甾醇類物質(zhì)一般提取之后會采取色譜分離法或者毛細(xì)管電泳法進(jìn)行純化。常用的色譜有柱色譜、薄層色譜和高效液相色譜等。Mallavadhani等[32]通過正己烷索氏提取和硅膠柱層析從草菇中分離出4個(gè)甾體代謝產(chǎn)物。李向敏[30]21-33通過硅膠柱層析和反相半制備液相色譜法從皺蓋假芝分離純化出麥角甾醇和麥角甾-7,22-二烯-3β-醇。徐俊蕾[33]通過甲醇浸膏提取、硅膠柱層析和凝膠柱層析等一系列分離純化，從松乳菇子實(shí)體中分離出少量麥角甾醇過氧化物。柱色譜設(shè)備簡單但是耗時(shí)耗力，需大量溶劑。薄層色譜操作簡單但是分離得到的產(chǎn)物較難回收。高效液相色譜法(HPLC)可進(jìn)行有效的分離，操作條件溫和，簡便高效，應(yīng)用廣泛。

3 甾醇的生理活性研究

3.1 抗炎作用

蘑菇中麥角甾醇抗炎活性研究進(jìn)展見表1。

3.2 其他生理活性

大量的研究表明麥角甾醇還具有抗腫瘤、抑菌、免疫調(diào)節(jié)[47]、抗病毒[48]和抗氧化[49]等多種活性。麥角甾醇具有抑制血管生長、激活抑癌基因或者通過細(xì)胞毒性直接殺死腫瘤細(xì)胞[30]12-17[50]的作用，是藥食用真菌發(fā)揮抑癌作用的主要小分子成分。而麥角甾醇顯著的抑菌活性[51]，也使蘑菇可以作為新抗菌劑的替代來源[9]。其他研究也表明麥角甾醇具有免疫調(diào)節(jié)作用[52]，能夠增強(qiáng)免疫力。除此之外，麥角甾醇作為維生素D的前體物質(zhì)，在紫外光照射下會轉(zhuǎn)化為維生素D2，因此食用菌也可以作為豐富的天然維生素D來源[53]。

4 甾醇的抗炎機(jī)理

4.1 炎癥反應(yīng)機(jī)制

炎癥是機(jī)體應(yīng)對損傷、刺激和感染的一種生理反應(yīng)。它與糖尿病、關(guān)節(jié)炎、癌癥和心血管疾病等多種疾病的發(fā)病機(jī)制相關(guān)[12,42,54]。巨噬細(xì)胞、單核細(xì)胞和其他炎癥細(xì)胞，在免疫刺激下會分泌許多炎癥介質(zhì)，如白細(xì)胞介素(IL-1β，IL-6，IL-8)、腫瘤壞死因子(TNF-α)、核因子κB(NF-κB)、誘導(dǎo)型環(huán)氧合酶-2(COX-2)、前列腺素E2(PGE2)和誘導(dǎo)型一氧化氮合酶(iNOS)，這些炎癥介質(zhì)大量分泌會啟動(dòng)多條信號轉(zhuǎn)導(dǎo)通路，引發(fā)炎癥，造成各種細(xì)胞損傷[12,55-56]。

4.2 發(fā)病機(jī)制

另一種重要的促炎介質(zhì)是由T淋巴細(xì)胞、肥大細(xì)胞、單核細(xì)胞和其他防御細(xì)胞所分泌的腫瘤壞死因子-α(TNF-α)[60]。當(dāng)存在免疫刺激時(shí)，TNF-α?xí)街谔囟ǖ目缒な荏w上，激活多條信號轉(zhuǎn)導(dǎo)通路，使得在感染部位生成更多TNF-α[61]。TNF-α不斷積累，造成廣泛的細(xì)胞凋亡、過度疼痛和細(xì)胞損傷。通過檢測被LPS激活的THP-1單核細(xì)胞或RAW 264.7巨噬細(xì)胞中TNF-α的抑制情況可以評價(jià)各種物質(zhì)抗炎效果[62]。

NF-κB通路是TNF-α調(diào)解過程中的關(guān)鍵過程。如圖3所示，巨噬細(xì)胞膜上的Toll樣受體(TLRs)和腫瘤壞死因子受體(TNFr)能夠識別多種病原相關(guān)分子模式(PAMPs)，激活髓樣分化蛋白88(MyD88)，MyD88又能激活絲裂原活化蛋白激酶(MAPK)，MAPK激活I(lǐng)KK激酶(IKKα、IKKβ、IKKγ)，導(dǎo)致IKB蛋白復(fù)合體的磷酸化[63]。IKB和NF-κB復(fù)合體降解使得NF-κB轉(zhuǎn)移至細(xì)胞核，NF-κB會引發(fā)多種參與免疫應(yīng)答反應(yīng)的因子翻譯，釋放大量炎癥介質(zhì)，引發(fā)炎癥。因此， NF-κB的天然抑制劑可以用于治療和預(yù)防各種炎癥疾病[64]。

表1 蘑菇中甾醇抗炎活性研究Table 1 Steroids isolated from mushrooms with reported anti-inflammatory activity

圖3 NF-κB炎癥機(jī)制圖示Figure 3 Schematic diagram of nuclear factor-κB (NF-κB) pathway

環(huán)氧合酶有2種亞型(COX-1和COX-2)，COX-1主要調(diào)節(jié)體內(nèi)平衡作用，促進(jìn)多種生理功能，COX-2受到刺激后會明顯表達(dá)，有助于炎癥的發(fā)展[65]。非選擇性非甾體類抗炎藥(NSAIDs)常用于治療疼痛和炎癥，但是由于其非選擇性對COX-1產(chǎn)生抑制作用會導(dǎo)致例如胃腸道、腎臟、高血壓和心血管毒性等多方面的藥物不良反應(yīng)[66]。而特異性抑制COX-2的藥物就可以既發(fā)揮抗炎作用又避免因抑制COX-1而產(chǎn)生副作用。

4.3 抗炎方法

目前的研究顯示菌菇中麥角甾醇的抗炎機(jī)理主要是抑制NO生成，減少TNF-α、IL-1β、IL-6、IL-12等細(xì)胞因子的釋放，抑制iNOS、COX2的表達(dá)，抑制NF-κB的活性以及減輕水腫。下面將對每一種機(jī)制的研究方法做簡單介紹。

4.3.1 一氧化氮試驗(yàn)(Griess反應(yīng)) Griess反應(yīng)是一項(xiàng)廣泛運(yùn)用于NO檢測和定量分析的簡單技術(shù)，基本反應(yīng)是磺胺和N-(1-萘基)乙二胺(NED)形成穩(wěn)定的偶氮化合物，這種化合物在540 nm處的吸光度與樣品中亞硝酸鹽的濃度呈正比。該方法經(jīng)許多研究者驗(yàn)證有效，是目前應(yīng)用最廣泛的測定抗炎活性的方法[12,34,40,56]。

4.3.2 細(xì)胞因子酶聯(lián)免疫吸附試驗(yàn)(ELISA) 酶聯(lián)免疫吸附試驗(yàn)用于細(xì)胞內(nèi)蛋白質(zhì)分泌和釋放的定性定量分析。這種方法通常用于細(xì)胞因子和其他炎癥介質(zhì)的定量分析[58,67]。通常將RAW 264.7細(xì)胞通過不同濃度樣品進(jìn)行篩選。最后收集細(xì)胞培養(yǎng)上清液根據(jù)ELISA試劑盒的操作說明進(jìn)行檢測來確定細(xì)胞中釋放的TNF-α和IL-6的量。

4.3.3 COX-1和COX-2催化的前列腺素生物合成試驗(yàn) 環(huán)氧合酶已經(jīng)廣泛應(yīng)用于天然物質(zhì)潛在抗炎作用的研究中[39,43]。RAW264.7細(xì)胞用LPS刺激誘導(dǎo)產(chǎn)生COX-2和其他炎癥介質(zhì)，不同濃度樣品處理后加入花生四烯酸進(jìn)一步培養(yǎng)，用PGE2酶免疫試劑盒檢測培養(yǎng)基中釋放的PGE2。

4.3.4 體內(nèi)試驗(yàn) 在小鼠體內(nèi)誘導(dǎo)炎癥并測量相對腫脹程度來評價(jià)天然產(chǎn)物的體內(nèi)抗炎活性。這些動(dòng)物模型都存在毛細(xì)血管擴(kuò)張，血管通透性增加以及與人類急性炎癥腫脹相類似的水腫等癥狀[68]?？刹扇「骨蛔⑸銵PS，10 h后處死取臟器檢測[36]?；蚪遣娌四z鹽溶液注射爪部，對照組用非甾體類抗炎藥進(jìn)行治療，不同的時(shí)間間隔測量爪部的體積增長[34,37]。

5 結(jié)論

食用菌種類多樣，資源豐富，且含有多種生理活性物質(zhì)，從中篩選具有抗炎作用的代謝產(chǎn)物潛力巨大。麥角甾醇作為菌類中活性物質(zhì)，在已有的研究中表現(xiàn)出優(yōu)良的抗炎作用。文獻(xiàn)整理過程中，發(fā)現(xiàn)蘑菇活性成分的提取方法多樣，但是中國還沒有蘑菇麥角甾醇提取測定的權(quán)威標(biāo)準(zhǔn)，這為相關(guān)領(lǐng)域的研究帶來些許不便。探究食用菌中麥角甾醇的提取分離方法，深入研究其抗炎作用機(jī)制對于天然產(chǎn)物抗炎藥物的篩選具有積極的意義。

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