摘要:沸水煮提貴州產(chǎn)天冬中的多糖成分并提純;測(cè)定其對(duì)·O2-和·OH的清除作用。結(jié)果顯示貴州產(chǎn)天冬中多糖的提取率為13.00%;一定劑量的天冬多糖在體外對(duì)·O2-和·OH具有顯著的清除作用(P<0.05)。
關(guān)鍵詞:天冬;多糖;提??;抗氧化
中圖分類號(hào):R284.2;O636.1 文獻(xiàn)標(biāo)識(shí)碼:A 文章編號(hào):0439-8114(2012)07-1436-02
Extraction of Polysaccharide from Guizhou Radix Asparagi and Its
Andioxidation Activity
LI Yan-ju1,LI Qin-shan2,TIAN Shuo2,LIU Yang3
(1. Hematology Blood Family, Affiliated Hospital of Guiyang Medical College, Guiyang 550004,China;
2. Teaching and Research Section of Biochemistry, Peking Union Medical College, Beijing 100730,China;
3. The College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022,China)
Abstract: Polysaccharide was extracted by boiling water method from Guizhou Radix Asparagi. The scavenging effects of Radix Asparagi polysaccharide on ·O2- and ·OH were determined. Results showed that the yield of Radix Asparagi polysaccharide was 13.00%. And the ·O2- and ·OH scavenging effects of Radix Asparagi polysaccharide at a certain dose were significant(P<0.05).
Key words: Radix Asparagi; polysaccharide; extraction; antioxidation;
天冬(Radix Asparagi)為百合科植物天門冬(Asparagus cochinchinensis(Lour.)Merr.)的干燥塊根[1]。國內(nèi)外研究發(fā)現(xiàn)天冬含有豐富的多糖和寡糖,具有類激素、抗氧化、增強(qiáng)免疫力、抗炎、抗腫瘤等多種作用[2-7],本試驗(yàn)以貴州產(chǎn)天冬為研究對(duì)象,提取和純化其中的多糖,并對(duì)其體外抗氧化活性進(jìn)行初步探討,為天冬的開發(fā)利用提供參考。
1 材料與方法
1.1 儀器和試劑
中藥天冬購于貴州鳳崗,3,5-二羥基甲苯購自中國藥品生物制品鑒定所,其余試劑均為國產(chǎn)分析純。
1.2 試驗(yàn)方法
1.2.1 天冬多糖的提取與純化[8] 稱取干燥的貴州天冬粉末120 g,加蒸餾水2 000 mL,沸水煮提6 h,過濾,殘?jiān)偌诱麴s水1 500 mL,煮提4 h。合并兩次提取液,濃縮至約200 mL,加體積分?jǐn)?shù)95%的乙醇使含醇量達(dá)到80%,沉淀多糖,靜置過夜。傾出上清液,抽濾,用60 mL體積分?jǐn)?shù)80%的乙醇洗滌3次得粗多糖。將粗多糖加熱溶于1 000 mL水中,用200 mL氯仿-正丁醇(體積比為4∶1)萃?。创?,除去氯仿層。母液加熱濃縮至200 mL,加體積分?jǐn)?shù)95%的乙醇使含醇量達(dá)80%,靜置過夜、抽濾、沉淀,依次用體積分?jǐn)?shù)95%的乙醇、無水乙醇、丙酮、乙醚洗滌,80 ℃干燥4 h,得精制天冬多糖,以上方法制備天冬多糖樣品6份,產(chǎn)品稱重后計(jì)算其得率。
1.2.3 天冬多糖抗氧化能力的測(cè)定
1)·O2-清除率的測(cè)定[9]。9 mL的PBS 50 mmoL/L(pH 8.34)、聯(lián)苯三酚0.2 mmoL/L,反應(yīng)溫度為25 ℃,分別加入0.2、0.4、0.8 g天冬多糖提取物,以不加多糖提取物的處理作對(duì)照,以pH 8.34的PBS作為參比,測(cè)定反應(yīng)啟動(dòng)后10 s的A322 nm。清除率按下式計(jì)算:
·O2-清除率=(A0-A1)÷A0×100%
式中,A0和A1分別表示對(duì)照和添加多糖處理的A322 nm。
2)·OH清除率的測(cè)定[10]。鄰二氮菲0.75 mmol/L、FeSO4 9.75 mmol/L、pH 7.34的PBS 150 μmol/L,加入質(zhì)量分?jǐn)?shù)0.01%的H2O2,37 ℃保溫60 min,加入0.40 g提取的天冬多糖,總反應(yīng)體系為10 mL。以未添加H2O2和天冬多糖的處理為陰性對(duì)照,以只添加H2O2的處理為空白對(duì)照,以同濃度多糖作參比,測(cè)定A536 nm。
·OH清除率=(A2-A0)/A0×100%
式中,A0、A2分別表示對(duì)照和添加多糖處理的A536 nm。
天冬多糖抗氧化能力試驗(yàn)中每項(xiàng)處理均設(shè)置6個(gè)重復(fù),試驗(yàn)結(jié)果均以x±s表示,并進(jìn)行差異顯著性分析。
2 結(jié)果與分析
2.1 沸水煮提法提取天冬中多糖的得率
按照1.2.1的方法提取天冬多糖,平均每120 g干燥的天冬粉末中可提取得到15.6 g精制天冬多糖,得率為13.00%,RSD=1.36%。
2.2 天冬多糖的抗氧化能力
2.2.1 天冬多糖對(duì)·O2-的清除作用 加入多糖后,反應(yīng)體系的A322 nm降低,表明天冬多糖對(duì)·O2-具有清除作用(表1)。在試驗(yàn)濃度范圍內(nèi),溶液的A322 nm隨天冬多糖添加量的增加而下降,當(dāng)天冬多糖添加量為0.4 g或0.8 g時(shí),反應(yīng)體系的A322 nm與不加多糖的空白對(duì)照相比有顯著差異(P<0.05),·O2-清除率分別高達(dá)19.32%和31.24%。
2.2.2 天冬多糖對(duì)·OH的清除作用 在本試驗(yàn)條件下,由A536 nm的測(cè)定值判斷天冬多糖對(duì)·OH的清除作用(表2),與陰性對(duì)照相比,只添加H2O2的處理A536nm顯著升高(P<0.05),而同時(shí)添加了H2O2和天冬多糖的處理A536 nm沒有顯著變化,表明天冬多糖對(duì)·OH有顯著的清除能力,清除率高達(dá)56.38%。
3 討論
隨著自由基研究的不斷深入,氧自由基造成的組織損傷已為人們所關(guān)注。它能破壞脂類、細(xì)胞膜、核酸及染色體,影響蛋白質(zhì)性質(zhì)及酶的活性[11-13]。試驗(yàn)證明當(dāng)機(jī)體內(nèi)·O2-及·OH過多時(shí),會(huì)產(chǎn)生大量脂質(zhì)過氧化物(LPO),損傷細(xì)胞膜,引起高血脂、冠心病、加速衰老等[14-18]。天冬為傳統(tǒng)中藥之一,在中國資源豐富,且具有多種潛在的藥用價(jià)值,具有抗菌,抗腫瘤等生物活性。前人報(bào)道,中藥天冬中含粘液質(zhì)、寡糖等成分[19,20]。本試驗(yàn)從貴州產(chǎn)天冬中提取多糖的得率為13.00%,且體外試驗(yàn)表明,達(dá)到一定劑量的天冬多糖對(duì)·O2-和·OH具有明顯的清除作用,從而推測(cè)天冬多糖在防治心血管疾病、抗衰老、防輻射等方面的作用可能與其能清除·O2-、·OH有關(guān),本試驗(yàn)可為貴州產(chǎn)天冬的開發(fā)與應(yīng)用奠定基礎(chǔ)。
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