基于液質(zhì)聯(lián)用技術(shù)的蟬花和冬蟲夏草中主要核苷類成分的比較及蟬花質(zhì)量標(biāo)準(zhǔn)研究
發(fā)布時(shí)間：2018-07-22 13:45
【摘要】：蟬花,為麥角菌科真菌大蟬草Cordyceps cicadae Shing的無性型蟬擬青霉Paecilomyces cicadae(Miq.)Samson寄生在山蟬Cicada flammata Dist幼蟲上的真菌孢梗束或子座及幼蟲尸體的干燥復(fù)合體。冬蟲夏草,為麥角菌科真菌冬蟲夏草菌Cordyceps sinensis(Ber K.)Sacc.寄生在蝙蝠蛾科昆蟲幼蟲上的子座和幼蟲尸體的干燥復(fù)合體�！吨袊幍洹穼Χx夏草有收錄,蟬花收錄于地方標(biāo)準(zhǔn)《四川省中藥材標(biāo)準(zhǔn)》中。蟬花主要產(chǎn)于江蘇、浙江、福建、四川、西藏等地,而冬蟲夏草主要分布于青海、西藏、四川、云南、甘肅和貴州等省及自治區(qū)。蟬花和冬蟲夏草同屬于麥角菌科,并且具有相似的藥理作用�，F(xiàn)代醫(yī)學(xué)研究表明,蟬花和冬蟲夏草均具有治療腎衰、增強(qiáng)免疫功能、抗疲勞、抗腫瘤等藥理活性。本研究建立了快速、靈敏的HPLC-MS/MS法同時(shí)測定了不同產(chǎn)地的20批蟬花和12批冬蟲夏草藥材中13種核苷類成分的含量,并同時(shí)測定了10批蟬花和6批冬蟲夏草的幼蟲和子實(shí)體中的含量,比較各成分在兩種藥材不同部位的異同。該實(shí)驗(yàn)通過比較蟬花和冬蟲夏草中主要核苷類成分的含量以及核苷類成分在兩種蟲草不同藥用部位的含量,初步探討了蟬花替代冬蟲夏草的可行性。目前,只有《四川省中藥材標(biāo)準(zhǔn)》對蟬花的質(zhì)量標(biāo)準(zhǔn)進(jìn)行了收載,且內(nèi)容只包括性狀、鑒別和檢查項(xiàng)。為對蟬花的質(zhì)量控制提供更可靠的手段,本文按照《中國藥典》2015年版的規(guī)范要求,從性狀、鑒別(薄層)、檢查(水分、總灰分、酸不溶性灰分和浸出物)、特征圖譜、含量測定等方面完善了蟬花的質(zhì)量標(biāo)準(zhǔn)。第一部分基于液質(zhì)聯(lián)用技術(shù)的蟬花和冬蟲夏草中主要核苷類成分的比較目的:建立HPLC-MS/MS法同時(shí)測定蟬花和冬蟲夏草中13中核苷類成分的含量,并同時(shí)比較同一藥材不同藥用部位的核苷類含量差異。方法:采用COSMOSIL Packed Column 5C18-PAQ(4.6×250 mm,5μm)色譜柱,以甲醇-0.05%甲酸水為流動相,梯度洗脫,流速為0.8 mL/min,進(jìn)樣量20μL,柱溫40℃。質(zhì)譜檢測采用多反應(yīng)監(jiān)測(MRM)模式,ESI源,正離子模式。源噴射電壓(IS)為5500 V;霧化溫度為650℃;霧化氣(GS1,N_2):4.14×10~5 Pa;輔助氣(GS2,N_2):4.48×10~5 Pa;氣簾氣(N_2):1.72×10~5 Pa。應(yīng)用Analyst Software 1.6.2軟件進(jìn)行定量分析數(shù)據(jù)的處理。結(jié)果:13種核苷類成分的峰面積與濃度在測定濃度范圍內(nèi)線性關(guān)系良好,相關(guān)系數(shù)R2≥0.9990。方法的精密度、準(zhǔn)確度、穩(wěn)定性、檢測限和定量限均符合要求,平均加樣回收率為95.9-103.4%。樣品測定結(jié)果表明,不同產(chǎn)地的蟬花和冬蟲夏草中13種核苷類成分的相對含量和絕對含量差別很大,并且冬蟲夏草中13種核苷類成分的總含量明顯高于蟬花,但兩種藥材中的含量最高的成分均為尿苷。通過比較兩種蟲草不同藥用部位即幼蟲和子實(shí)體中13種成分的總含量發(fā)現(xiàn),無論在蟬花還是冬蟲夏草中都是子實(shí)體中的含量大于幼蟲中的含量。結(jié)論:首次建立了簡便、靈敏度高、專屬性強(qiáng)的HPLC-MS/MS法,用于同時(shí)測定蟬花和冬蟲夏草中13種核苷類成分的含量,并比較了兩種蟲草不同藥用部位中核苷類成分含量差異,為蟬花替代冬蟲夏草的可行性提供了一定的理論基礎(chǔ)。第二部分蟬花質(zhì)量標(biāo)準(zhǔn)研究目的:建立蟬花質(zhì)量標(biāo)準(zhǔn),為不同來源蟬花藥材的全面質(zhì)量控制提供依據(jù)。方法:通過查閱文獻(xiàn)對蟬花進(jìn)行本草考證;依據(jù)《中國藥典》2015年版四部通則,并參考《四川省中藥材標(biāo)準(zhǔn)》對10批蟬花進(jìn)行性狀、鑒別(薄層色譜鑒別)、檢查(水分、總灰分、酸不溶性灰分、醇溶性浸出物)、特征圖譜和含量測定等方法的建立。結(jié)果:1.確定了蟬花的基源和性狀。2.鑒別:薄層色譜鑒別可見5個(gè)顯色清晰的主斑點(diǎn),分離度達(dá)到要求,并且該分離條件受溫度和濕度影響較小。3.檢查:水分測定結(jié)果為6.1%~11.6%;總灰分測定結(jié)果為5.5%~15.5%;酸不溶性灰分測定結(jié)果為2.0%~10.5%;浸出物測定結(jié)果為39.4%~45.6%。4.特征圖譜:建立了蟬花的特征圖譜。5.含量測定:尿苷的質(zhì)量濃度在2.68~16.13μg/m L范圍內(nèi)與峰面積線性關(guān)系良好,回歸方程為Y=0.833X-0.084(R2=0.9999),加樣回收率為98.30%~101.4%,尿苷的含量范圍為0.095%~0.172%。結(jié)論:建立了專屬性和耐用性良好的薄層色譜鑒別方法;制定了水分、總灰分、酸不溶性灰分、醇溶性浸出物的限度;建立了簡便、穩(wěn)定的特征圖譜和含量測定方法,可用于蟬花藥材的質(zhì)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284.1;O657.63
本文編號：2137656
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284.1;O657.63
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