鐘靜詩(shī), 張 健, 王共明, 劉 芳, 劉 奎, 王藝欣, 劉海超, 趙云蘋

海參皂苷的制備及結(jié)構(gòu)解析研究進(jìn)展

鐘靜詩(shī)1, 2, 張 健2, 王共明2, 劉 芳2, 劉 奎1, 2, 王藝欣1, 2, 劉海超1, 2, 趙云蘋2

(1.上海海洋大學(xué) 食品學(xué)院, 上海 201306; 2. 山東省海洋資源與環(huán)境研究院, 山東 煙臺(tái) 264006)

海參皂苷是海參體內(nèi)的一種次生代謝產(chǎn)物, 主要用于抵御捕食者的侵害, 具有有效的抗腫瘤、抗帕金森病、免疫調(diào)節(jié)等作用, 應(yīng)用前景廣泛。提取制備海參皂苷是其他研究的基礎(chǔ), 而分析結(jié)構(gòu)與活性的關(guān)系有助于深入理解其多種生物活性效應(yīng)產(chǎn)生的原因。本文著重對(duì)目前海參皂苷的提取、分離純化方法及結(jié)構(gòu)分析進(jìn)行了概述, 并對(duì)海參皂苷制備方法的優(yōu)化和結(jié)構(gòu)與生物活性之間的關(guān)系進(jìn)行了分析展望, 以期能為相關(guān)研究提供參考。

海參皂苷; 提取分離; 皂苷結(jié)構(gòu)

海參, 又稱海黃瓜, 屬棘皮動(dòng)物, 體型呈橢圓柱狀, 個(gè)體柔軟。海參主要分為海參綱和刺身綱, 在我國(guó)沿海地區(qū)廣泛存在。全球有超過1 716種海參的存在, 其中大部分分布在亞洲地區(qū)[1]。目前較多學(xué)者研究的海參品種主要有[2](刺參屬)、[3](海參屬)、[4](白尼參屬)、[5](瓜參屬)、[6](輻肛參屬)和[7](梅花參屬)。

皂苷是由糖基和配基組成的一類糖苷, 其苷元為三萜或螺旋甾烷類化合物[8], 在植物如人參、桔梗、遠(yuǎn)志、甘草、柴胡中比較常見[9], 而在動(dòng)物中發(fā)現(xiàn)較少。海洋生物皂苷主要存在于海參、海星等海洋生物體內(nèi)[10]。海參具有與其他棘皮動(dòng)物不同的特點(diǎn), 它們能產(chǎn)生皂苷以抵御捕食者和寄生生物[11], 但目前尚不清楚海參產(chǎn)生皂苷具體的分子機(jī)制[12]。

目前, 對(duì)海參皂苷的研究主要集中在提取、分離純化及活性研究方面。根據(jù)海參皂苷具有“親水親油”的特點(diǎn), 通常采用溶劑提取法提取, 采用沉淀法和色譜法等手段進(jìn)行分離純化。在海參皂苷的生物活性方面, 相關(guān)學(xué)者研究發(fā)現(xiàn)海參皂苷具有抗腫瘤[13]、抗帕金森病[14]、進(jìn)行免疫調(diào)節(jié)[15]等多種生物活性。但相較于海參其他生物活性物質(zhì), 海參皂苷的研究報(bào)道尚少, 且對(duì)于皂苷分子結(jié)構(gòu)與其活性之間的構(gòu)效關(guān)系研究相對(duì)缺乏。對(duì)海參皂苷的提取優(yōu)化及分子結(jié)構(gòu)的深入研究, 將有助于探明海參皂苷生物活性的分子基礎(chǔ), 并促進(jìn)其活性的開發(fā)和應(yīng)用。本文對(duì)海參皂苷的提取、分離純化、結(jié)構(gòu)及理化性質(zhì)進(jìn)行了綜述, 以期為海參皂苷的進(jìn)一步研究提供參考。

1 海參皂苷的提取

1.1 海參皂苷的提取來源

海參皂苷是海參的次生代謝產(chǎn)物, 在體壁、消化腺、生殖腺、呼吸樹和居維氏管中都發(fā)現(xiàn)了皂苷的存在[16-19]。目前較多學(xué)者研究海參體壁、消化腺和居維氏管中的皂苷。袁文鵬等[19]從海參體壁、消化腺中提取海參皂苷進(jìn)行營(yíng)養(yǎng)成分的對(duì)比。Chumphoochai等[20]從海參體壁和居維氏管中提取海參皂苷并對(duì)其活性進(jìn)行研究。研究表明海參體壁皂苷含量不及海參內(nèi)臟中的皂苷[19], 但相較于海參體壁, 海參內(nèi)臟皂苷的研究較少。

除了從海參中提取皂苷外, 海參燙漂后得到的廢水也是皂苷的來源之一。海參用熱水燙漂能使海參自溶酶失活, 阻止海參的自溶, 但這也導(dǎo)致了一些營(yíng)養(yǎng)物質(zhì)如蛋白質(zhì)、氨基酸、碳水化合物、礦物質(zhì)、皂苷的喪失。Yin等[21]發(fā)現(xiàn)利用熱水處理海參會(huì)損失大量海參體壁中的皂苷, 損失量約41%。有研究報(bào)道稱海參和海參燙漂液中都含有海參皂苷, 海參中皂苷含量約為0.81%, 而海參燙漂液中皂苷含量約為0.68%[22]。

1.2 海參皂苷的提取方法

皂苷通?？扇苡谒痛既芤? 不溶于乙醚、氯仿和苯。根據(jù)皂苷性質(zhì)對(duì)海參進(jìn)行提取, 常見的提取方法為醇溶液提取和在此基礎(chǔ)上改善使用大孔樹脂進(jìn)行提取。兩種提取方法的流程如圖1所示。

圖1 常見海參皂苷提取方法流程圖

醇提取法常用提取液有甲醇、乙醇、正丁醇等。除此之外, 有學(xué)者使用乙酸乙酯或多種溶劑按一定配比混合提取海參皂苷。目前, 提取海參皂苷使用較多的溶劑為乙醇, 而60%和70%乙醇體系最為常見, 推測(cè)與海參皂苷結(jié)構(gòu)具有“兩親性”, 采用60%～70%乙醇相較于其他濃度乙醇提取海參皂苷的效率更高有關(guān)。

大孔吸附樹脂法和醇提取法的區(qū)別在于大孔樹脂法采用大孔吸附樹脂代替了石油醚、乙醚和氯仿的萃取。相關(guān)研究認(rèn)為, 大孔樹脂提取法提取效率較高且能有效地去除混雜的鹽類物質(zhì)[23]。如表1所示, 在提取仿刺參屬皂苷時(shí), 利用大孔樹脂法提取得總皂苷20.5 mg·g–1, 明顯高于醇提取法的得率。另外, 對(duì)海參的預(yù)處理有利于提升海參皂苷的得率。有報(bào)道稱在使用復(fù)合酶對(duì)海參進(jìn)行酶解預(yù)處理后, 海參皂苷得率提升了一倍[24], 這為海參皂苷提取提供新思路。常見海參皂苷的提取方法具體見表1。

1.3 海參皂苷的含量測(cè)定

目前關(guān)于海參皂苷測(cè)定方法的研究尚少, 測(cè)定其他皂苷的常用方法有比色法、薄層色譜法和高效液相色譜法等。比色法具有簡(jiǎn)便、快捷的優(yōu)點(diǎn), 但其準(zhǔn)確度會(huì)受到其他雜質(zhì)的干擾; 薄層色譜法利用一定展開劑展開, 再聯(lián)合運(yùn)用其他儀器進(jìn)行定量測(cè)定; 高效液相色譜法能有效的分離樣品和雜質(zhì), 在皂苷的分離和測(cè)定中較常使用。薄層色譜法和高效液相色譜法都需標(biāo)準(zhǔn)品進(jìn)行對(duì)照, 然而目前海參皂苷尚不具有高品質(zhì)標(biāo)準(zhǔn)品, 對(duì)海參皂苷檢測(cè)方法的選擇造成一定的局限性。因此, 對(duì)海參粗皂苷進(jìn)行含量測(cè)定時(shí), 常采用比色法, 而對(duì)單個(gè)或幾個(gè)單體皂苷的含量測(cè)定多使用后兩種方法。比色法中使用的標(biāo)準(zhǔn)品除了海參皂苷單體外, 還可采用其他植物皂苷或齊墩果酸, 而高效液相色譜法則多采用分離純化出的海參皂苷。不同方法測(cè)定海參皂苷含量通常可改變顯色劑、色譜柱等以提高海參皂苷含量測(cè)定的準(zhǔn)確性, 部分海參皂苷含量的測(cè)定方法詳見表2。

海參皂苷不具有發(fā)色團(tuán), 采用紫外分光光度法無法直接對(duì)其進(jìn)行含量測(cè)定。因此, 運(yùn)用比色法對(duì)海參皂苷含量進(jìn)行測(cè)定時(shí), 需要通過溶劑與皂苷相互作用產(chǎn)生生色團(tuán)從而在一定波長(zhǎng)內(nèi)檢測(cè)出皂苷的含量。由表2知, 比色法常使用香草醛-高氯酸或香草酸-冰醋酸的顯色體系顯色。香草醛-酸顯色體系的原理可能是海參皂苷在酸的作用下水解使C3位置上的羥基能與香草醛發(fā)生羥醛縮合反應(yīng)而顯色。楊志文等[37]在研究人參皂苷的定量分析中也持有相同的觀點(diǎn)。

2 海參皂苷的分離純化

通常提取后的海參皂苷為粗皂苷, 包含了多種單體皂苷。為了進(jìn)一步研究海參皂苷活性及海參皂苷活性與海參單體皂苷之間的關(guān)系, 需對(duì)海參粗皂苷進(jìn)行分離純化。

海參皂苷多數(shù)為三萜皂苷, 少數(shù)為螺旋甾烷類皂苷。三萜類化合物分離的傳統(tǒng)方法主要有分段沉淀法、膽甾醇沉淀法。分段沉淀法使用乙醚、丙醇或其混合物對(duì)不同極性海參皂苷進(jìn)行分離; 膽甾醇沉淀法利用膽甾醇飽和的醇溶液和乙醚分離純化海參皂苷。分段沉淀法和膽甾醇沉淀法的流程如圖2所示。

表1 海參皂苷部分提取方法

注: —: 無對(duì)應(yīng)項(xiàng)目。下同。

表2 部分海參皂苷含量測(cè)定方法

圖2 海參皂甙分離純化流程圖

分段沉淀法和膽甾醇沉淀法的區(qū)別在于分段沉淀法是通過使用梯度溶劑逐步分離極性不同的皂苷。而膽甾醇沉淀法則是利用膽甾醇先與皂苷結(jié)合, 再通過去除膽甾醇得到純化的海參皂苷。三萜皂苷通常不與甾醇形成穩(wěn)定的復(fù)合物[38], 因此, 膽甾醇沉淀法還可用于三萜皂苷和甾體皂苷的分離。

色譜法是目前分離海參皂苷最常用的方法。用于海參皂苷分離的色譜法主要有大孔吸附色譜法、硅膠色譜法、高效液相色譜法和薄層層析色譜法等。以上方法主要根據(jù)皂苷的極性的不同對(duì)海參皂苷進(jìn)行分離。其中大孔吸附色譜柱的方法主要是通過水洗和梯度的醇溶液的洗滌, 分離出糖及水溶性雜質(zhì)和依次洗脫出極性不同的皂苷。吸附劑的不同決定了該種層析法不同的吸附特性[39]; 硅膠色譜法通常采用氯仿-甲醇-水混合溶液進(jìn)行梯度洗脫, 其特點(diǎn)是能分離分離度較大的化合物[40]。高效液相色譜法在分離純化皂苷中最常見, 常用的色譜柱為ODS色譜柱, 洗脫劑為甲醇-水或甲醇。薄層色譜法分離純化海參皂苷則常用甲醇-水或乙醇進(jìn)行分離純化。色譜柱、流動(dòng)相和洗脫液的選擇影響海參皂苷的分離純化的效果, 部分海參皂苷分離純化方法見表3。

表3 部分海參皂苷分離純化方法

凝膠色譜法是根據(jù)分子量進(jìn)行分離的一種層析方法, 海參皂苷分子量的大小主要取決于糖苷部分。凝膠色譜法在海參皂苷的分離純化中應(yīng)用的相關(guān)報(bào)道較少, 利用凝膠色譜法分子篩原理可分離具有不同分子量大小的海參皂苷。由表3可以看出, 為達(dá)到分離純化的目的, 通常采用多種分離方法結(jié)合以得到較純海參皂苷。

高速逆流色譜是近年來逐步發(fā)展的一項(xiàng)新技術(shù),它與傳統(tǒng)的色譜層析主要的不同點(diǎn)在于它是利用液-液萃取對(duì)提取物進(jìn)行分離, 即用于分離的固定相和流動(dòng)相都為液體。利用高效逆流色譜進(jìn)行分離能避免固相對(duì)提取物的不可逆吸附, 并且它對(duì)樣品的要求不高, 粗提物就可直接進(jìn)行分離。目前已有研究報(bào)道高速逆流色譜法用于黃酮[47]、生物堿[48]、醌類[49]、香豆木脂素[50]等的分離純化。近年來, 高速逆流色譜用于皂苷的分離純化主要集中于柴胡[51]、薯蕷[52]、人參[53]等中藥材, 用于海參皂苷的分離純化較少。

3 海參皂苷的結(jié)構(gòu)分析

海參皂苷具有幾種特征性的理化性質(zhì), 據(jù)此可以判斷提取純化的化合物是否為皂苷。海參皂苷在水溶液中震蕩能產(chǎn)生持久性的泡沫, 具有發(fā)泡性, 原因是海參皂苷是由親水性的糖鏈和親脂性的苷元組成的表面活性劑。皂苷鑒定最常用的顯色反應(yīng)為L(zhǎng)ibermann-Burchar反應(yīng), 又稱醋酐濃硫酸反應(yīng)。若在兩層界面之間有紫紅色反應(yīng), 則說明有苷類物質(zhì)的存在。Molish反應(yīng)也能判斷提取物中是否含有苷類物質(zhì), 其判斷依據(jù)是液面之間是否存在紫紅色環(huán)[49]。

根據(jù)苷元部分的不同可將海參皂苷分為螺旋甾烷類皂苷和三萜類皂苷。截至2015年, 已經(jīng)發(fā)現(xiàn)的海參皂苷的種類有700余種[44], 其中大多數(shù)為三萜皂苷。三萜類皂苷多數(shù)由30個(gè)C組成, 通常認(rèn)為皂苷由六個(gè)異戊二烯單位聚合而成, 且多數(shù)為四環(huán)三萜或五環(huán)三萜。海參皂苷還可以分為海參烷型皂苷和非海參烷型皂苷, 它們的區(qū)別在于海參烷型皂苷含有18(20)-內(nèi)酯環(huán)而非海參烷型皂苷含有18(16)-內(nèi)酯環(huán)或不含有內(nèi)酯環(huán)[54-56]。

組成海參皂苷糖基部分的單糖主要有D-葡萄糖(Glc), D-木糖(Xyl), D-奎諾糖(Qui), 3-O-甲基-D-葡萄糖(MeGlc)和3-O-甲基-D-木糖[55], 皂苷寡糖鏈通常由2-6個(gè)單糖組成[54]。

海參皂苷苷元母核結(jié)構(gòu)式及糖鏈常見單糖結(jié)構(gòu)式如圖3所示。

圖3 海參皂苷母核及其糖基部分常見單糖結(jié)構(gòu)式

注: a. 海參烷性皂苷母核; b. 非海參烷型皂苷母核; c. D-葡萄糖; d. D-木糖; e. D-奎諾糖; f. 3-O-甲基-D-葡萄糖; g. 3-O-甲基-D-木糖

海參皂苷糖苷中單糖上是否含有硫酸基團(tuán)以及硫酸基團(tuán)所處的位置(如木糖C-4位、葡萄糖C-6位、奎諾糖C-3位)、海參皂苷配基及配基側(cè)鏈中是否含有雙鍵以及雙鍵的數(shù)量和位置(如7(8)、9(11)、24(25)、25(26)、位上的雙鍵)、苷元中羥基、環(huán)氧基、乙?；?主要在配基的C-16、C-22、C-23和或C-25上[56])等的數(shù)量和類型[54]都使得海參皂苷具有多樣性。

另外, Van Dyck等[57]研究發(fā)現(xiàn)海參皂苷之間能發(fā)生相互轉(zhuǎn)化, 他們分析了在掠食者應(yīng)激和未受干擾的條件下對(duì)皂苷的影響, 發(fā)現(xiàn)兩種狀態(tài)下皂苷的種類不同, 有的皂苷會(huì)發(fā)生轉(zhuǎn)化形成另一種皂苷。這與Kamyab等[25]的觀點(diǎn)一致。

常用的理化性質(zhì)只能對(duì)海參皂苷是否存在進(jìn)行簡(jiǎn)單判斷, 通過現(xiàn)代儀器分析技術(shù)能進(jìn)一步探究海參皂苷單體的結(jié)構(gòu)。

海參皂苷是苷元為三萜類或螺旋甾烷類化合物的一種糖苷。采用傅里葉紅外變換[45]、質(zhì)譜[58]和核磁共振[59]等技術(shù)能解析海參皂苷的結(jié)構(gòu)。紅外光譜能通過分子振動(dòng)產(chǎn)生的吸收頻率判斷化合物所含基團(tuán); 核磁共振能根據(jù)原子核的化學(xué)位移判斷化合物的結(jié)構(gòu); 質(zhì)譜能根據(jù)化合物的分子離子峰判斷出化合物的相對(duì)分子質(zhì)量, 再根據(jù)質(zhì)荷比確定分子式, 最后通過離子峰確定化合物的結(jié)構(gòu)。質(zhì)譜在定性方面應(yīng)用廣泛, 通過聯(lián)用氣相色譜或液相色譜, 能同時(shí)對(duì)化合物進(jìn)行定量分析。

4 展望

目前, 海參皂苷的研究尚處于起步階段, 海參皂苷的提取分離仍缺乏統(tǒng)一且高效的方法, 建立海參皂苷制備的方法是研究海參皂苷的重要且基礎(chǔ)性難題。大多數(shù)學(xué)者采用醇溶液提取海參皂苷, 化學(xué)試劑用量大, 對(duì)環(huán)境污染也較大。表面活性劑具有短時(shí)、低成本、優(yōu)化有效成分、綠色環(huán)保等特點(diǎn)[60], 已應(yīng)用于三七皂苷、薯蕷皂苷、人參皂苷等植物皂苷的提取研究中[61-63]。探究表面活性劑等友好型溶劑提取海參皂苷成為研究海參皂苷制備一個(gè)新方向。

另外, 海參皂苷的不同活性及活性強(qiáng)弱與海參皂苷的分子結(jié)構(gòu)有關(guān), 已有研究表明海參皂苷中硫酸基團(tuán)、羰基對(duì)海參皂苷活性有影響[64]。因此, 單體皂苷的分離純化及其結(jié)構(gòu)解析、單體皂苷結(jié)構(gòu)與活性之間的關(guān)系仍是當(dāng)前海參皂苷研究重點(diǎn)和難點(diǎn)之一。下一步, 采用新型溶劑體系或表面活性劑提取海參皂苷, 利用超聲、微波、酶解等現(xiàn)代科學(xué)新技術(shù)輔助提取, 優(yōu)化海參皂苷的提取工藝; 采用多種方法相結(jié)合及應(yīng)用高速逆流色譜法等新方法促進(jìn)海參單體皂苷的分離純化; 探究皂苷分子結(jié)構(gòu)與活性的關(guān)系, 皂苷結(jié)構(gòu)中的功能基團(tuán)如含硫酸基團(tuán)、含氧基團(tuán)等對(duì)活性的影響等都是急需解決的研究?jī)?nèi)容。

近年來, 隨著國(guó)內(nèi)外學(xué)者對(duì)海參皂苷的深入研究, 其多種生物活性如抗腫瘤、抗帕金森和免疫調(diào)節(jié)等逐漸被人們認(rèn)知。繼續(xù)研究海參皂苷豐富的生物學(xué)活性, 明確各種活性的分子生物學(xué)機(jī)制, 對(duì)針對(duì)性的開發(fā)相關(guān)功能性產(chǎn)品, 高值化開發(fā)利用皂苷, 對(duì)保障人類健康, 提升人們的生活質(zhì)量都具有重要作用。

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Research progress in the preparation and structural analysis of sea cucumber saponins

ZHONG Jing-shi1, 2, ZHANG Jian2, WANG Gong-ming2, LIU Fang2, LIU Kui1, 2, WANG Yi-xin1, 2, LIU Hai-chao1, 2, ZHAO Yun-ping2

(1. College of Food Sciences & Technology, Shanghai Ocean University, Shanghai 201306, China; 2. Shandong Marine Resource and Environment Research Institute, Yantai 264006, China)

Sea cucumber saponins, which are mainly used for defense against predators, are secondary metabolites with anti-tumor, anti-Parkinson’s disease, and immune-regulating bioactivities. These also have broad potential applications. The extraction and preparation of sea cucumber saponins are the basis of other studies, and the analysis of the relationship between their structures and activities is helpful for an in–depth understanding of their multiple biological activity effects. In this study, the extraction, separation, purification, and structural analysis of sea cucumber saponins are summarized. Furthermore, the optimization of the preparation of sea cucumber saponins and the relationship between their structures and biological activities are prospected to provide a reference for related research.

sea cucumber saponins; extraction and separation; structure of saponins

Sep. 12, 2020

TS201.2

A

1000-3096(2022)04-0133-10

10.11759/hykx20200912002

2020-09-12;

2021-04-16

山東省現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系刺參產(chǎn)業(yè)創(chuàng)新團(tuán)隊(duì)建設(shè)項(xiàng)目(SDAIT-22-07)

[Construction Program Stichopus japonicus Industrial Innovation Team of Modern Agricultural Industrial Technology System of Shandong Province, No. SDAIT-22-07]

鐘靜詩(shī), (1997—), 女, 廣東惠州人, 碩士研究生, 主要從事食品工程研究, E-mail: 465369582@qq.com; 張健(1980—),通信作者, 山東臨沂人, 博士, 副研究員, 主要從事海洋食品科學(xué)研究, E-mail: zjsd408@163.com

(本文編輯: 康亦兼)