白术多糖硫酸酯制备工艺优化及抗乳腺增生作用

厉昌旭; 李烁; 张意可; 戚泽筠; 汤威威; 高琪; 刘嘉悦; 邵梦婷; 王艳艳; 赵宏

doi:10.13386/j.issn1002-0306.2022100083

白术多糖硫酸酯制备工艺优化及抗乳腺增生作用

Optimization of Preparation Technology and Anti-Mammary Gland Hyperplasia of Polysaccharide Sulfate from Atractylodes macrocephala

摘要

摘要: 目的：建立一种具有抗乳腺增生作用的白术多糖（Atractylodes macrocephala Koidz polysaccharide，AMP）硫酸酯的制备方法。方法：采用浓硫酸法对AMP进行硫酸化修饰（sulfated polysaccharides from Atractylodes macrocephala Koidz，SAMP），通过响应面法确定硫酸酯化反应最佳工艺条件；采用高效凝胶渗透色谱法（HPGPC）、红外光谱法（IR）对AMP及SAMP的结构进行初步鉴定；通过测定AMP与SAMP对DPPH、ABTS⁺、OH自由基清除能力，考察其体外抗氧化活性；通过苯甲酸雌二醇-黄体酮联用建立大鼠MGH模型，记录大鼠体重及乳房直径，观察大鼠乳腺病理组织形态。结果：最佳工艺条件为反应试剂比（浓硫酸:正丁醇）=2:1，反应温度−4 ℃，反应时间21 min，在此条件下SAMP取代度为0.59±0.00391。HPGPC测定结果显示AMP、SAMP分子质量分别为2719、33524 Da；红外光谱显示SAMP在833、1226 cm⁻¹出现C-O-S和S=O特征吸收峰，表明AMP修饰成功；当多糖溶液质量浓度为0.5 mg/mL时，SAMP对DPPH、ABTS⁺、OH自由基清除率分别为54.91%、38.21%、42.23%。同模型组比较，SAMP组大鼠乳头直径减小（P<0.01），乳腺腺泡数量减少，乳腺增生症状减轻，且SAMP治疗效果优于AMP。结论：响应面法优化AMP硫酸酯化工艺方法稳定可行，且AMP经硫酸酯化修饰后，具有更好的抗乳腺增生作用。

Abstract: Objective: To establish a preparation method of Atractylodes macrocephala Koidz polysaccharide (AMP) sulfate with anti-hyperplasia of mammary. Methods: AMP were sulfated by sulfated method, and the optimal conditions for the sulfated modification were determined by response surface methodology. The structures of AMP and SAMP were initially analyzed by infrared spectroscopy (IR) and High-Performance Gel Chromatography (HPGPC). The anti-oxidative activity in vitro of polysaccharide was investigated by testing the DPPH, ABTS⁺, OH free radical scavenging ability of AMP and SAMP. Rat model of mammary gland hyperplasia was established by intramuscular injection of estradiol benzoate and progesterone. The rats weight and breast diameter were recorded and the pathological organization form of mammary gland of rats were observed. Results: The optimum preparation conditions were as follows: Ratio of conditions of volume ratio of sulfuric acid and n-butanol 2:1, reaction temperature −4 ℃, reaction time 21 min. The degree of substitution of SAMP was 0.59±0.00391. The HPGPC results showed the relative molecular weights for AMP and SAMP were 2719 and 33524 Da, respectively. The IR spectra showed that SAMP had characteristic absorption peaks of S=O and C-O-S appeared near 833 and 1226 cm⁻¹, indicating that the sulfated was successful. When the sample concentration was 0.5 mg/mL, the DPPH, ABTS⁺, OH free radical scavenging activities of SAMP were 54.91% , 38.21%, and 42.23%, respectively. Compared with the model group, the diameter of the nipples of the rats in the SAMP group decreased (P<0.01), the number of acinars were decreased, the mammary gland hyperplasia was reduced and SAMP exhibits stronger anti MGH activity than AMP. Conclusion: The study indicated that the optimized processing technology of SAMP modified by response surface methodology was stable and feasible and the sulfated modification could increase the anti-MGH capacity of AMP.