不同分子量龙须菜多糖对草酸钙结晶的调控作用

植物多糖的化学结构与尿液中的结石抑制剂葡胺聚糖相似，有可能用于预防和治疗肾结石。天然多糖由于分子量和分子体积过大，导致其应用受到限制。研究了四种分子量分别为49.6，16.2，8.2和3.8 kDa的降解龙须菜多糖GLP1，GLP2，GLP3和GLP4对草酸钙(CaOx)晶体生长的调控作用。1H NMR，13C NMR和气相色谱-质谱(GC-MS)谱分析表明四种GLPs由β-D-半乳糖和6-O-硫酸基-3,6-α-L-吡喃半乳糖组成。X射线衍射(XRD)检测表明，在各GLPs存在下，诱导了二水草酸钙(COD)晶体形成，COD的衍射峰出现在晶面间距d=0.617，0.441，0.277和0.224 nm处；而没有多糖存在时只生成一水草酸钙(COM)晶体，COM的衍射峰出现在d=0.593，0.364，0.296和0.235 nm。由于COD比COM更容易排出体外，COD的形成有利于降低结石形成的风险。傅里叶变换红外光谱(FTIR)检测表明，随着GLP分子量减小或GLP浓度增加，草酸根中羧基的不对称伸缩振动ν_as(COO-)和对称对称伸缩振动ν_s(COO-)都发生了不同程度的蓝移，其中ν_as(COO-)从1 618 cm-1增加到1 642 cm-1，ν_s(COO-)从1 318 cm-1增加到1 328 cm-1，即GLP4诱导的全部是COD晶体。扫描电子显微镜(SEM)检测表明，随着GLP分子量减小，不但晶体中COD的比例增加，而且晶体的分散程度增大，晶体更加圆钝。随着GLP分子量减小或GLP浓度增加，其诱导生成的CaOx晶体表面电荷越负，Zeta电位绝对值越大，这有利于抑制晶体的聚集。电感耦合等离子体发射光谱仪(ICP)检测表明，四种GLPs均可以增加溶液中可溶性Ca2+的浓度，同时减少CaOx沉淀的生成量。在浓度为1.0 g·L-1多糖存在时，上清液中可溶性Ca2+的摩尔浓度分别为：GLP4 (37.88 μmol·L-1)>GLP3 (19.70 μmol·L-1)>GLP2 (16.05 μmol·L-1)>GLP1 (10.55 μmol·L-1)。结果表明，四种GLPs均可以抑制COM生长，诱导COD生成，降低晶体的聚集程度，增加晶体表面的Zeta电位绝对值和溶液中可溶性Ca2+浓度，减少CaOx晶体的生成量，且GLPs的调控活性与其分子量呈负相关。这些结果提示GLPs特别是分子量最小的GLP4有可能是防治CaOx结石的潜在药物。

Effects of Different Molecular Weight of Gracilaria LemaneiformisPolysaccharide on Calcium Oxalate Crystal

Plant polysaccharides’chemical structures are similar to that of glucosaminoglycan,a stone inhibitor in urine;thus plant polysaccharides may be used to prevent and treat kidney stones.However,the applications of natural polysaccharides are limited due to their large molecular weights and volume.In this study,the effects of four degraded Gracilaria lemaneiformis polysaccharides(GLP1,GLP2,GLP3 and GLP4)with molecular weights of 49.6,16.2,8.2 and 3.8 kDa,respectively,on crystal growth of calcium oxalate(CaOx)were studied.1H NMR,13 C NMR and gas chromatography-mass spectrometry(GC-MS)spectra showed that the four GLPs were composed ofβ-D-galactose and 6-O-sulfate-3,6-α-L-galactopyranose.X-ray diffraction(XRD)patterns showed that GLPs induced the formation of calcium oxalate dihydrate(COD)crystals,and the diffraction peaks of COD appeared at crystal plane spacing d=0.617,0.441,0.277 and 0.224 nm.However,only calcium oxalate monohydrate(COM)crystals were formed in the absence of polysaccharide,and the diffraction peaks of COM appeared at d=0.593,0.364,0.296 and 0.235 nm.Because COD is more easily excreted out from the body than COM,COD formation is beneficial to reduce the risk of stone formation.Fourier-transform infrared spectroscopy(FTIR)showed that the asymmetric stretching vibrationνas(COO-)and symmetric stretching vibrationνs(COO-)of carboxyl groups from oxalate had different degrees of blue shift as the molecular weight of GLP decreased or GLP concentration increased,in whichνas(COO-)increased from 1618 to 1642 cm^-1andνs(COO-)increased from 1318 to 1328 cm^-1.That is,GLP4 induced full COD crystals.Scanning electron microscope(SEM)examination showed that as the molecular weight of GLP decreases,the proportion of COD increased,and the dispersion degree of the crystal increased,making the crystal much blunter.As GLP molecular weight decreased or GLP concentration increased,the surface charges of CaOx crystals induced by GLPs changed to be more negative.The greater absolute value of Zeta potential is conducive to inhibiting crystal aggregation.The inductively coupled plasma emission spectra(ICP)results revealed that the four GLPs increased the concentration of soluble Ca^( 2+)ions(c(Ca^( 2+)))in the solution and decreased the amount of CaOx precipitation.At a concentration of 1.0 g·L^-1,the c(Ca^( 2+))in the supernatant were GLP4(37.88μmol·L^-1)>GLP3(19.70μmol·L^-1)>GLP2(16.05μmol·L^-1)>GLP1(10.55μmol·L^-1),respectively.The four GLPs can inhibit COM growth,induce COD formation,reduce the aggregation degree of crystals,increase the absolute value of Zeta potential on crystal surface and the concentration of soluble Ca^( 2+)ions in solution,and reduce the amount of CaOx crystal formation.The regulatory ability of GLP was negatively correlated with its molecular weight.All these results showed that GLPs,especially GLP4 with the smallest molecular weight,might be potential drugs for the preventing and treating CaOx stones.