[關(guān)鍵詞]
[摘要]
目的 從海藻中制備并分離純化一種低相對(duì)分子質(zhì)量海藻多糖(LMSP),初步分析其結(jié)構(gòu)特征并研究其抗氧化、降血糖和抗腫瘤作用。方法 水提醇沉法制備海藻粗多糖,H2O2/維生素C(VC)法降解海藻多糖,經(jīng)DEAE-52陰離子交換色譜柱和葡聚糖凝膠G-100色譜柱分離純化;采用苯酚-硫酸法測(cè)定總糖含量,間羥聯(lián)苯法測(cè)定糖醛酸含量,BaCl2-明膠比濁法測(cè)定硫酸基含量,考馬斯亮藍(lán)法測(cè)定蛋白質(zhì)含量,高效凝膠滲透色譜測(cè)定相對(duì)分子質(zhì)量,進(jìn)行紅外光譜法分析,高效液相色譜測(cè)定單糖組成,氣相色譜-質(zhì)譜聯(lián)用儀檢測(cè)糖苷鍵類型,掃描電鏡分析形貌特征;1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率實(shí)驗(yàn)、羥自由基清除率實(shí)驗(yàn)、2,2-聯(lián)氮-二(3-乙基-苯并噻唑-6-磺酸)二銨鹽(ABTS)自由基清除率實(shí)驗(yàn)檢測(cè)LMSP抗氧化活性;α-淀粉酶抑制實(shí)驗(yàn)、α-葡萄糖苷酶抑制實(shí)驗(yàn)檢測(cè)LMSP降血糖活性;檢測(cè)LMSP對(duì)A549、HeLa、Hep-G2細(xì)胞的抗腫瘤活性。結(jié)果 LMSP的重均相對(duì)分子質(zhì)量為4 635,總糖質(zhì)量分?jǐn)?shù)為(83.85±2.12)%,糖醛酸的質(zhì)量分?jǐn)?shù)為(29.73±3.57)%,硫酸根質(zhì)量分?jǐn)?shù)為(24.44±1.97)%,蛋白質(zhì)的質(zhì)量分?jǐn)?shù)為(1.35±0.63)%,主要由半乳糖、木糖和巖藻糖組成,紅外光譜及氣相色譜-質(zhì)譜聯(lián)用對(duì)其結(jié)構(gòu)分析推測(cè),該多糖可能是一種含吡喃糖苷的硫酸多糖,主要含有2-Rhap、T-Galp、3-Galp和4-Glcp等片段,LMSP在掃描電鏡下呈現(xiàn)出帶孔的網(wǎng)狀結(jié)構(gòu),堆疊緊密。LMSP對(duì)DPPH自由基清除率、羥基自由基清除率和ABTS自由基清除率的半數(shù)抑制濃度(IC50)為1.35、0.61、1.31 mg·mL-1;對(duì)α-淀粉酶和α-葡萄糖苷酶的IC50分別為0.82、1.27 mg·mL-1;對(duì)A549、HeLa和Hep-G2細(xì)胞的IC50為2.31、3.41、3.10 mg·mL-1,且各指標(biāo)的作用均強(qiáng)于海藻粗多糖。結(jié)論 H2O2/VC法降解制備得到均一的LMSP,具有良好的抗氧化、降血糖和抗腫瘤作用。
[Key word]
[Abstract]
Objective To isolate and purify a low molecular weight Sargassum polysaccharides (LMSP) from Sargassum of traditional Chinese medicine, and preliminarily analyze its structural characteristics and study its antioxidant, hypoglycemic and antitumor effects. Methods The seaweed polysaccharides were extracted by water extraction and ethanol precipitation, and then degraded by H2O2/VC method. They were purified by DEAE-52 anion exchange column and Sephadex G-100 gel filtration column. The total sugar content was determined by phenol-sulfuric acid method, the sugarfarn acid content was determined by p-hydroxybenzaldehyde method, the sulfate content was determined by BaCl2-gelatin turbidimetry method, and the protein content was determined by Coomassie brilliant blue method. The relative molecular mass was determined by high-performance gel permeation chromatography, the infrared spectrum was analyzed, and the composition of monosaccharides was determined by highperformance liquid chromatography. The type of glycosidic bond was detected by gas chromatography-mass spectrometry. The morphological features were analyzed by scanning electron microscopy. The antioxidant activity of LMSP was determined by 1,1- diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging rate experiment, hydroxyl radical scavenging rate experiment, and 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging rate experiment. The hypoglycemic activity of LMSP was determined by α -amylase inhibition experiment and α -glucosidase inhibition experiment. The antitumor activity of LMSP against A549, Hela, and Hep-G2 cells was determined. Results The relative molecular mass of LMSP is 4 635, with a total sugar content of (83.85±2.12)% and a sugar aldonic acid content of (29.73±3.57)%. The sulfate content is (24.44±1.97)%, the protein content is (1.35±0.63)%, and it is mainly composed of galactose, xylose, and fucose. Infrared spectroscopy and gas chromatography-mass spectrometry were used to analyze its structure, and it was inferred that LMSP may be a pyransulfonic polysaccharide containing pyransulfonic glycosides, mainly containing 2-Rhap, T-Galp, 3-Galp, and 4-Glcp fragments. Under scanning electron microscope, LMSP presents a porous network structure, tightly stacked. The half maximal inhibitory concentration (IC50) for DPPH radical scavenging, hydroxyl radical scavenging, and ABTS radical scavenging were 1.35, 0.61, and 1.31 mg·mL-1, respectively. The IC50 values for α-amylase and α-glucosidase inhibition were 0.82 and 1.27 mg·mL-1, respectively. The IC50 values for A549, HeLa, and Hep-G2 cell inhibition were 2.31, 3.41, and 3.10 mg·mL-1, respectively, and the effects of all indicators were stronger than those of seaweed crude polysaccharides. Conclusion The LMSP prepared by the H2O2/VC method is homogeneous and has good antioxidant, glucose-lowering, and anti-tumor effects.
[中圖分類號(hào)]
R284.2
[基金項(xiàng)目]
國家科技部重點(diǎn)研發(fā)項(xiàng)目(2016YFC0800908);國家科技部重大研究專項(xiàng)(2014ZX09509);山東省重點(diǎn)研發(fā)項(xiàng)目(2017CXGC1305)