基于液质联用技术的蝉花和冬虫夏草中主要核苷类成分的比较及蝉花质量标准研究

发布时间：2018-07-22 13:45

【摘要】：蝉花,为麦角菌科真菌大蝉草Cordyceps cicadae Shing的无性型蝉拟青霉Paecilomyces cicadae(Miq.)Samson寄生在山蝉Cicada flammata Dist幼虫上的真菌孢梗束或子座及幼虫尸体的干燥复合体。冬虫夏草,为麦角菌科真菌冬虫夏草菌Cordyceps sinensis(Ber K.)Sacc.寄生在蝙蝠蛾科昆虫幼虫上的子座和幼虫尸体的干燥复合体。《中国药典》对冬虫夏草有收录,蝉花收录于地方标准《四川省中药材标准》中。蝉花主要产于江苏、浙江、福建、四川、西藏等地,而冬虫夏草主要分布于青海、西藏、四川、云南、甘肃和贵州等省及自治区。蝉花和冬虫夏草同属于麦角菌科,并且具有相似的药理作用。现代医学研究表明,蝉花和冬虫夏草均具有治疗肾衰、增强免疫功能、抗疲劳、抗肿瘤等药理活性。本研究建立了快速、灵敏的HPLC-MS/MS法同时测定了不同产地的20批蝉花和12批冬虫夏草药材中13种核苷类成分的含量,并同时测定了10批蝉花和6批冬虫夏草的幼虫和子实体中的含量,比较各成分在两种药材不同部位的异同。该实验通过比较蝉花和冬虫夏草中主要核苷类成分的含量以及核苷类成分在两种虫草不同药用部位的含量,初步探讨了蝉花替代冬虫夏草的可行性。目前,只有《四川省中药材标准》对蝉花的质量标准进行了收载,且内容只包括性状、鉴别和检查项。为对蝉花的质量控制提供更可靠的手段,本文按照《中国药典》2015年版的规范要求,从性状、鉴别(薄层)、检查(水分、总灰分、酸不溶性灰分和浸出物)、特征图谱、含量测定等方面完善了蝉花的质量标准。第一部分基于液质联用技术的蝉花和冬虫夏草中主要核苷类成分的比较目的:建立HPLC-MS/MS法同时测定蝉花和冬虫夏草中13中核苷类成分的含量,并同时比较同一药材不同药用部位的核苷类含量差异。方法:采用COSMOSIL Packed Column 5C18-PAQ(4.6×250 mm,5μm)色谱柱,以甲醇-0.05%甲酸水为流动相,梯度洗脱,流速为0.8 mL/min,进样量20μL,柱温40℃。质谱检测采用多反应监测(MRM)模式,ESI源,正离子模式。源喷射电压(IS)为5500 V;雾化温度为650℃;雾化气(GS1,N_2):4.14×10~5 Pa;辅助气(GS2,N_2):4.48×10~5 Pa;气帘气(N_2):1.72×10~5 Pa。应用Analyst Software 1.6.2软件进行定量分析数据的处理。结果:13种核苷类成分的峰面积与浓度在测定浓度范围内线性关系良好,相关系数R2≥0.9990。方法的精密度、准确度、稳定性、检测限和定量限均符合要求,平均加样回收率为95.9-103.4%。样品测定结果表明,不同产地的蝉花和冬虫夏草中13种核苷类成分的相对含量和绝对含量差别很大,并且冬虫夏草中13种核苷类成分的总含量明显高于蝉花,但两种药材中的含量最高的成分均为尿苷。通过比较两种虫草不同药用部位即幼虫和子实体中13种成分的总含量发现,无论在蝉花还是冬虫夏草中都是子实体中的含量大于幼虫中的含量。结论:首次建立了简便、灵敏度高、专属性强的HPLC-MS/MS法,用于同时测定蝉花和冬虫夏草中13种核苷类成分的含量,并比较了两种虫草不同药用部位中核苷类成分含量差异,为蝉花替代冬虫夏草的可行性提供了一定的理论基础。第二部分蝉花质量标准研究目的:建立蝉花质量标准,为不同来源蝉花药材的全面质量控制提供依据。方法:通过查阅文献对蝉花进行本草考证;依据《中国药典》2015年版四部通则,并参考《四川省中药材标准》对10批蝉花进行性状、鉴别(薄层色谱鉴别)、检查(水分、总灰分、酸不溶性灰分、醇溶性浸出物)、特征图谱和含量测定等方法的建立。结果:1.确定了蝉花的基源和性状。2.鉴别:薄层色谱鉴别可见5个显色清晰的主斑点,分离度达到要求,并且该分离条件受温度和湿度影响较小。3.检查:水分测定结果为6.1%~11.6%;总灰分测定结果为5.5%~15.5%;酸不溶性灰分测定结果为2.0%~10.5%;浸出物测定结果为39.4%~45.6%。4.特征图谱:建立了蝉花的特征图谱。5.含量测定:尿苷的质量浓度在2.68~16.13μg/m L范围内与峰面积线性关系良好,回归方程为Y=0.833X-0.084(R2=0.9999),加样回收率为98.30%~101.4%,尿苷的含量范围为0.095%~0.172%。结论:建立了专属性和耐用性良好的薄层色谱鉴别方法;制定了水分、总灰分、酸不溶性灰分、醇溶性浸出物的限度;建立了简便、稳定的特征图谱和含量测定方法,可用于蝉花药材的质量控制。
[Abstract]:An asexual Paecilomyces cicadae (Miq.) Samson parasitic on the fungal peduncle of the cicada Cicada flammata Dist larva and the dry complex of the corpse and the corpse of the larvae of Cordyceps cicadae Shing. C. parasitic on the dry complex of the sons and larvae of the larvae of the family moth. < Chinese Pharmacopoeia > included in the Cordyceps sinensis. Cicadas are included in the local standard < Sichuan traditional Chinese medicine standard >. Cicadas are mainly produced in Jiangsu, Zhejiang, Fujian, Sichuan, Tibet and other places, and the winter insect and summer grass are mainly distributed in Qinghai, Tibet, Sichuan, Yunnan, Gansu. The cicadas and Cordyceps sinensis belong to the family of ergodaceae and have similar pharmacological effects. Modern medical studies have shown that cicadas and Cordyceps are both effective in the treatment of renal failure, enhancing immune function, anti fatigue, and anti-tumor activity. The rapid and sensitive HPLC-MS/MS method has been established for the simultaneous determination of different HPLC-MS/MS methods. The content of 13 nucleosides in the 20 batches of cicadas and 12 batches of Cordyceps sinensis, and the content of the 10 batches of cicadas and 6 batches of Cordyceps, and the similarities and differences in the different parts of the two kinds of medicinal materials were compared. The content of the main nucleoside components in cicadas and the winter worm and summer grass were compared. The content of glycosides in different medicinal parts of two species of Cordyceps, preliminary discussion on the feasibility of cicadas substitutes for Cordyceps sinensis. At present, only the standard of Chinese medicinal materials in Sichuan province has been carried out, and the contents include only characters, identification and examination. The standard requirement of the national pharmacopoeia >2015 edition is that the quality standards of cicadas are perfected from characters, identification (thin layer), examination (water, total ash, acid insoluble ash and Leach), characteristic atlas and content determination. The first part is based on the comparison of the main nucleosides in cicadas and summer grass based on liquid chromatography-use Technology: the establishment of HPLC-MS/MS The content of nucleosides in cicadas and Cordyceps sinensis 13 was measured simultaneously, and the difference of nucleoside contents in different medicinal parts of the same medicine was also compared. Methods: using COSMOSIL Packed Column 5C18-PAQ (4.6 x 250 mm, 5 m) column, methanol -0.05% formic water as the flow phase, gradient elution, flow rate of 0.8 mL/min, and sample volume 20 L, column The temperature was 40 degrees centigrade. Mass spectrometry was detected by multi reaction monitoring (MRM) model, ESI source and positive ion mode. Source injection voltage (IS) was 5500 V; atomization temperature was 650; atomization gas (GS1, N_2): 4.14 x 10~5 Pa; auxiliary gas (GS2, N_2): 4.48 x 10~5 Pa; 1.72 X: 1 The relationship between the peak area and concentration of the 3 nucleosides was good in determining the concentration range. The precision, accuracy, stability, detection limit and quantitative limit of the correlation coefficient R2 > 0.9990. were all conformed to the requirements. The average recovery rate of 95.9-103.4%. samples showed that 13 nucleosides in cicadas and Cordyceps sinensis from different habitats The relative content and absolute content vary greatly, and the total content of 13 nucleosides in Cordyceps sinensis is obviously higher than that of cicada cicada, but the highest content of the two kinds of medicinal herbs is uridine. By comparing the total content of 13 species in the different medicinal parts of the two species of Cordyceps, namely the larvae and the fruiting bodies, they are found in the cicadas and the winter insects. It is the first time that the content of 13 nucleosides in cicadas and Cordyceps sinensis is determined by HPLC-MS/MS method, which is simple, sensitive and strong, and compares the difference of nucleoside content in the different medicinal parts of two kinds of Cordyceps, which is a substitute for the winter insect of cicadas. The second part of cicadas quality standard research objective: to establish the quality standard of cicadas, to provide the basis for the comprehensive quality control of cicadas in different sources. Methods: through the literature review of the cicadas, according to the four general rules of Chinese Pharmacopoeia >2015 edition, and reference to the traditional Chinese medicine in Sichuan Province. Material standard > for the 10 batch of cicadas, identification (TLC identification), examination (water, total ash, acid insoluble ash, alcohol soluble extract), the establishment of characteristics and content determination. Results: 1. determine the basis and character.2. identification of cicadas: TLC can identify 5 main spots with clear color, and the separation degree reaches the requirement. And the separation condition is affected by the temperature and humidity of small.3. examination: the result of water determination is 6.1%~11.6%; the result of determination of total ash is 5.5%~15.5%; the result of acid insoluble ash determination is 2.0%~10.5%; the determination result of the extract is 39.4%~45.6%.4. characteristic Atlas: the determination of the characteristic Atlas of the cicadas is.5. content determination: the mass concentration of uridine in 2.68~ There is a good linear relationship with peak area in the range of 16.13 g/m L, the regression equation is Y=0.833X-0.084 (R2=0.9999), the rate of addition recovery is 98.30%~101.4%, the content of uridine is 0.095%~0.172%. conclusion: the identification method of thin layer chromatography with good specificity and durability is established, and the water, total ash, acid insoluble ash and alcohol soluble extract are formulated. A simple and stable characteristic map and content determination method were established for the quality control of cicada flower.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284.1;O657.63

【参考文献】