Optimization of Ultrasonic-Microwave Assisted Extraction of Polysaccharides from Eucommia ulmoides Leaves and Its Anticoagulant Activity in Vitro

To develop the rich resources of Eucommia ulmoides leaves in China, the optimal conditions of extracting polysaccharides from Eucommia ulmoides leaves by ultrasonic and microwave, the physicochemical properties and the anticoagulant activity in vitro of the extracted polysaccharides were studied. Firstly, the ranges of factors affecting the yield of polysaccharide, such as ultrasonic power, extraction temperature, microwave power, ratio of solid to liquid, extraction time, were determined by single factor experiment. The key factors were further screened by Plackett­Burman test, and the Box-Behnken test was used to optimize the process conditions. Finally, the physicochemical properties, such as molecular weight and monosaccharide composition, and the anticoagulant indices, such as activated partial prothrombin time (APTT), prothrombin time (PT), thrombin time (TT) of the refined Eucommia ulmoides leaf polysaccharides, were analyzed. The results showed that the ratio of solid to liquid, the extraction time and the extraction temperature were the key factors. The optimal extraction conditions were the ultrasonic power of 130 W, the extraction temperature of 49 ℃, the microwave power of 200 W, the solid-liquid ratio of 1 to 30 (g:mL) and the extraction time of 20 min. Under these conditions, the actual yield of Eucommia ulmoides leaf polysaccharides was 4.02%±0.03%, close to the theoretical yield of 4.08%. The weight-average molecular mass (M_w) of the refined polysaccharide was 1653 kDa, and the composition and molar ratio of monosaccharide were 37.3% fructose, 35% glucose, 14.6% n-acetyl-d-glucosamine, 8.6% galactose and 4.4% arabinose. The results showed that the polysaccharides of Eucommia ulmoides leaves was β-type acidic polysaccharide. Compared with the negative control, the Eucommia ulmoides polysaccharides significantly increased APTT (P<0.01), and significantly increased PT and TT (P<0.01) at the concentration of 8 mg/mL, indicating that it mainly affected coagulation system through endogenous pathway, could also affect the coagulation process through exogenous and common pathways. This study could provide theoretical basis and technical reference for the high-value utilization of Eucommia ulmoides leaves in China.