健脾益肾丸指标成分的含量测定及其对CRF模型大鼠钙磷代谢和炎症因子的影响
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篇名: 健脾益肾丸指标成分的含量测定及其对CRF模型大鼠钙磷代谢和炎症因子的影响
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摘要: 目的:建立健脾益肾丸(JYP)中黄芪甲苷、大黄素、大黄酚的含量测定方法,并探讨JYP对慢性肾衰竭(CRF)模型大鼠钙磷代谢和炎症因子等相关指标的影响。方法:采用高效液相色谱法进行含量测定。黄芪甲苷以及大黄素、大黄酚的色谱柱分别为Agilent Zorbax SB-C18、Agilent TC C18,流动相分别为乙腈-水(36 ∶ 64,V/V)、甲醇-0.1%磷酸溶液(75 ∶ 25,V/V),检测器分别为蒸发光散射检测器和二极管阵列检测器(后者检测波长为254 nm),柱温分别为30、25 ℃,流速均为1.0 mL/min,进样量分别为20、10 μL。将SD大鼠随机分为正常组、模型组、尿毒清组(1.80 g/kg)和JYP低、中、高剂量组(1.71、3.43、6.85 g/kg),每组10只。除正常组外,其余组大鼠均采用5/6肾切除方法复制CRF模型。造模4个月后,正常组和模型组大鼠均灌胃相应体积水,各给药组大鼠灌胃相应药物,每日1次,连续12周。采用酶联免疫吸附测定法检测大鼠血肌酐(Scr)、尿素氮(BUN)、甲状旁腺激素(PTH)和炎症因子[白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)]含量,采用甲基百里香酚蓝比色法和磷钼酸法检测其血钙、血磷含量,采用Pearson检验考察炎症因子与钙磷代谢相关指标(血钙、血磷、PTH)的相关性。结果:黄芪甲苷、大黄素、大黄酚检测质量浓度的线性范围分别为54.537~381.759、2.960~20.720、6.318~44.223 μg/mL(r>0.999),定量限分别为0.010、0.288、0.216 μg/mL,检测限分别为0.003、0.096、0.072 μg/mL,精密度、重复性、稳定性试验的RSD均小于3.0%,加样回收率为97.18%~102.33%(RSD<3%,n=9)。造模后(给药前),模型组和各给药组大鼠血清Scr、BUN含量均较正常组显著升高(P<0.01);给药后,各给药组大鼠上述指标均较模型组和同组给药前显著降低(P<0.01)。与正常组比较,模型组大鼠血钙含量显著降低,IL-6、TNF-α含量均显著升高(P<0.01);与模型组比较,JYP中、高剂量组大鼠血钙含量均显著升高,尿毒清组PTH含量,JYP中、高剂量组PTH、IL-6含量以及各给药组TNF-α含量均显著降低(P<0.05或P<0.01);但JYP对大鼠血磷含量无显著影响,且其血清炎症因子与钙磷代谢相关指标均无显著相关性(P>0.05)。结论:本含量测定方法简便、专属性强、灵敏度高,可用于JYP中黄芪甲苷、大黄素、大黄酚的含量测定。JYP可改善CRF模型大鼠的肾功能,缓解其钙代谢紊乱,并抑制其炎症因子的表达。
ABSTRACT: OBJECTIVE: To establish the method for the content determination of astragaloside Ⅳ, emodin and chrysophanol in Jianpi yishen pills (JYP) and to investigate the effects of JYP on calcium, phosphorus metabolism and inflammatory factors in chronic renal failure (CRF) model rats. METHODS: HPLC method was adopted. The determination of astragaloside Ⅳ, emodin and chrysophanol was perform on Agilent Zorbax SB-C18, Agilent TC C18 column, respectively; mobile phase consisted of acetonitrile-water (36 ∶ 64, V/V) and methanol-0.1% phosphoric acid solution (75 ∶ 25, V/V); the detectors were evaporative light-scattering detector and diode-array detector (detection wavelength of 254 nm); the column temperatures were set at 30 ℃and 25 ℃ at the flow rate of 1.0 mL/min; the sample sizes were 20 and 10 μL. SD rats were randomly divided into normal group, model group, Niaoduqing group (1.80 g/kg) and JYP low-dose, medium-dose and high-dose groups (1.71, 3.43, 6.85 g/kg), with 10 rats in each group. Except for normal group, CRF model of other groups were established by 5/6 nephrectomy in other groups. Four months after modeling, normal group and model group were given constant volume of water intragastrically; admi- nistration groups were given relevant medicine intragastrically, once a day, for consecutive 12 weeks. The levels of serum creatinine (Scr), urea nitrogen (BUN), parathyroid hormone (PTH) and inflammatory factors (IL-6, TNF-α) were measured by ELISA. Methyl thymol blue colorimetric method and phosphomolybdic acid method were used to detect the contents of blood calcium and phosphorus. Correlation of inflammatory factors with related calcium and phosphorus metabolism indexes (blood calcium, blood phosphorus, PTH) were investigated with Pearson assay. RESULTS: The linear range of astragaloside Ⅳ, emodin and chrysophanol were 54.537-381.759, 2.960-20.720, 6.318-44.223 μg/mL, respectively. The limits of quantitation were 0.010, 0.288, 0.216 μg/mL; the limits of detection were 0.003, 0.096, 0.072 μg/mL. RSDs of precision, reproducibility and stability tests were all lower than 3.0%. The recoveries were 97.18%-102.33%(RSD<3%,n=9). After modeling (before medication), serum contents of Scr and BUN in model group and administration group were increased significantly, compared with normal group (P<0.01). After medication, above indexes of administration group were decreased significantly, compared with model group and the same group before medication (P<0.01). Compared with normal group, the content of blood calcium were decreased significantly, while the contents of IL-6 and TNF-α were increased significantly (P<0.01). Compared with model group, the content of blood calcium were increased significantly in JYP medium-dose and high-dose groups, while serum content of PTH in Niaoduqing group, serum contents of PTH and IL-6 in JYP medium-dose and high-dose groups as well as serum content of TNF-α in administration group were decreased significantly (P<0.05 or P<0.01). JYP had no significant effect on blood phosphorus in rats, and there was no correlation of inflammatory factors with related calcium and phosphorus metabolism indexes (P>0.05). CONCLUSIONS: The established content determination method is simple, specific and sensitive, and can be used for content determination of astragaloside Ⅳ, emodin and chrysophanol in JYP. JYP can improve renal function of CRF model rats, relieve calcium metabolism disorder and inhibit the expression of inflammatory factors.
期刊: 2019年第30卷第16期
作者: 江霞,陈秋谷,郭丽琴,胡兆流,黄诗莹,王佛长,郑平,易铁钢,张尚斌,李顺民,陈剑平
AUTHORS: JIANG Xia,CHEN Qiugu,GUO Liqin,HU Zhaoliu,HUANG Shiying,WANG Fochang,ZHENG Ping,YI Tiegang,ZHANG Shangbin,LI Shunmin,CHEN Jianping
关键字: 健脾益肾丸;黄芪甲苷;大黄素;大黄酚;含量测定;高效液相色谱法;慢性肾衰竭;钙磷代谢;炎症因子;大鼠
KEYWORDS: Jianpi yishen pills; Astragaloside Ⅳ; Emodin; Chrysophanol; Content determination; HPLC; Chronic renal failure; Calcium and phosphorus metabolism; Inflammatory factors; Rat
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