广金钱草多糖指纹图谱的建立、含量测定及其对α-葡萄糖苷酶的抑制活性研究

Fingerprint Establishment, Content Determination and α-glucosidase Inhibitory Activity Study of Polysaccharide from Desmodium styracifolium

目的:建立广金钱草多糖的指纹图谱,分析其单糖组成及含量,并考察广金钱草多糖对α-葡萄糖苷酶的体外抑制作用。方法:采用水提醇沉法提取广金钱草多糖,经三氟乙酸水解、1-苯基-3-甲基-5-吡唑啉酮衍生化后,采用高效液相色谱法(HPLC)建立指纹图谱(以葡萄糖峰为参照),并进行单糖组成及含量分析。含量测定色谱柱为Phenomenex Luna C18,流动相为乙腈-0.05mol/L磷酸二氢钾溶液(用氢氧化钠调节pH至6.8),梯度洗脱,流速为0.8 mL/min,检测波长为250 nm,柱温为30℃,进样量为10μL。以阿卡波糖为对照,采用对硝基苯-α-D-葡萄糖吡喃苷法考察广金钱草多糖对α-葡萄糖苷酶的体外抑制活性。结果:18批药材样品的HPLC指纹图谱有9个共有峰,其中15批药材样品的相似度大于0.90;共指认甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖等7个共有峰。其中,鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖的含量分别为0.471~2.092、1.379~8.919、2.560~35.679、1.194~6.905、0.566~4.158 mg/g;以鼠李糖为基准,其余4种单糖的摩尔比分别为1.58~4.07、2.26~19.95、2.20~4.21、1.31~2.86。广金钱草多糖对α-葡萄糖苷酶的抑制活性呈现随剂量升高而增强的趋势,其半数抑制浓度为0.70 mg/mL,低于阳性对照阿卡波糖(7.76 mg/mL)。结论:不同批次广金钱草的多糖组成均以葡萄糖为主,各单糖含量有所差异。广金钱草多糖对α-葡萄糖苷酶具有明显的体外抑制作用,且活性强于阿卡波糖。

OBJECTIVE:To establish the fingerprint,analyze the monosaccharide composition and content,investigate the inhibitory effects of the polysaccharide from Desmodium styracifolium on α-glucosidase in vitro. METHODS:Polysaccharide from D. styracifolium was prepared by water extraction and ethanol precipitation. After hydrolyzed by TFA and derived by PMP,HPLC method was adopted to establish the fingerprint(using glucose peak as reference),and analyze the constituent and content of monosaccharide. The content determination was performed on Phenomenex Luna C18 column with mobile phase consisted of acetonitrile-0.05 mol/L potassium phosphate(pH adjusted to 6.8 with sodium hydroxide)in gradient elution at the flow rate of 0.8 mL/min. The detection wavelength was set at 250 nm,and column temperature was set at 30 ℃. The sample size was 10 μL. Using acarbose as control,PNPG assay was used to investigate the α-glucosidase inhibitory activity of polysaccharide from D. styracifolium.RESULTS:There were 9 common peaks in HPLC fingerprints of 18 batches of samples,and the similarity of 15 batches of samples was higher than 0.90. Totally 7 peaks were identified as mannose,rhamnose,galacturonic acid,glucose,galactose,xylose and arabinose. The contents of rhamnose,galacturonic acid,glucose,galactose and arabinose were 0.471-2.092,1.379-8.919,2.560-35.679,1.194-6.905,0.566-4.158 mg/g,respectively. Based on rhamnose,the molar ratios of the other four monosaccharides were 1.58-4.07,2.26-19.95,2.20-4.21 and 1.31-2.86,respectively. The inhibitory activity of polysaccharide from D. styracifolium on α-glucosidase increased with the increase of dose,and the half inhibitory concentrations of it was 0.70 mg/mL,lower than 7.76 mg/mL of acarbose(positive control). CONCLUSIONS:Glucose is the main component of D. styracifolium polysaccharide in different batches,and the contents of monosaccharides are different. The polysaccharide from D. styracifolium have significant inhibitory activity on α-glucosidase,which is better than that of acarbose.