灵芝孢子粉多糖色谱指纹图谱及其免疫活性谱效关系的研究
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摘要
灵芝作为传统的食药两用真菌,在我国已有两千多年历史。随着近年来对灵芝中多糖、三萜、生物碱、腺苷和甾醇等成分的深入研究,灵芝的生理活性作用越来越受到人们的广泛关注。灵芝多糖作为灵芝中生理活性物质的杰出代表,随灵芝直接或被加工后以保健品、药品以及功能性食品的形式被推向市场而发挥奇效。然而伴随这些产品迅猛发展态势的却是质量良莠不齐、真假难辨的问题。鉴于多糖组成和结构的复杂性,目前仍缺乏能够科学有效地表征和鉴别灵芝及灵芝孢子粉中活性多糖成分的常规检测方法和质量标准。本课题以破壁灵芝孢子粉为实验材料,通过水浸超声辅助提取法提取灵芝多糖,采用TFA部分水解耦合PMP-HPLC的分析方法检测和表征不同灵芝孢子粉样品中多糖的组成特性,并建立灵芝孢子粉多糖的PMP-HPLC标准指纹图谱。此外,还采用酸-酶水解耦合离子色谱的方法,分别建立了灵芝孢子粉多糖酸-酶水解后单糖和寡糖组分的HPAEC标准指纹图谱;通过MTT实验研究灵芝孢子粉多糖对小鼠淋巴细胞增殖的促进效应,并初步考察了多糖指纹图谱与其免疫活性之间的谱效关系。
为了使指纹图谱更好地表征灵芝多糖的特征信息,本课题对灵芝多糖TFA部分水解的条件进行了考察。通过对TFA浓度、水解温度和水解时间进行单因素以及正交试验,以共有特征峰的峰面积和作为衡量指标,确定了最优水解条件:TFA浓度0.4 mol/L、水解温度110℃和水解时间70 min。
在上述水解条件下对15个灵芝孢子粉多糖样品进行PMP-HPLC分析,然后运用“中药色谱指纹图谱相似度评价系统2004A版”软件处理,建立了灵芝孢子粉多糖的PMP-HPLC标准指纹图谱,其中确定了19个共有特征峰,且共有峰峰面积和占总峰面积的95%以上。对于这15个样品指纹图谱相似度分析的结果表明,同一产地不同批次产品间的相似度很高,大于0.97;不同产地产品间的相似度较低,但均大于0.80。而对赤芝孢子粉以外的其它灵芝多糖类样品进行同样分析,则能够看到明显差异。
同样,为了得到理想的离子色谱指纹图谱,本课题对离子色谱分析条件进行了优化。在此条件下也对灵芝孢子粉多糖样品离子色谱分析前的酸-酶部分水解条件进行了考察。通过对硫酸浓度、水解温度和水解时间的单因素以及正交试验,综合考虑水解物中单糖和寡糖组分的共有特征峰峰面积和这两个指标后,确定了最优水解条件:硫酸浓度3 mol/L、水解温度90℃和水解时间2 h。在此条件下我们分别建立了灵芝孢子粉多糖水解产物单糖和寡糖组分的HPAEC标准指纹图谱。
为了考察灵芝孢子粉多糖的谱效关系,我们选择MTT实验表征灵芝孢子粉多糖的免疫活性。利用SPSS 17.0软件对20个样品MTT实验结果与其离子色谱指纹图谱中单、寡糖共有峰的相对峰面积数据进行多元回归分析,得到能够表征灵芝孢子粉多糖免疫活性的多元线性回归方程。
As one of the traditional fungi for both edible and medicinal use, Ganoderma lucidum has a history of more than two thousand years in China. With in-depth studies of components in Ganoderma lucidum such as polysaccharides, triterpenoids, alkaloids, adenosines and sterols, its physiological activities arrest more and more attention. Ganoderma lucidum polysaccharides, an outstanding representative of physiologically active substances among them, have played its important role as Ganoderma lucidum have come to market in the form of health care products, pharmaceuticals and functional food. Ganoderma lucidum and its products have mushroomed, while the quality of these products on the market is hard to distinguish, as well as to tell a true from a false. As the complexity of the composition and structure of polysaccharides, there is still a lack of analysis methods and quality standards which could effectively characterize and identify the polysaccharides from Ganoderma lucidum with physiological activity. In order to resolve the problem, we took the broken Ganoderma lucidum spore powder as the experimental material, and extracted Ganoderma lucidum polysaccharides by ultrasound-assisted extraction method. In our study, two methods of TFA partial acidic hydrolysis coupled PMP pre-column derivatization reverse phase high performance liquid chromatography (RP-HPLC) and acid-enzyme partial hydrolysis coupled high-performance anion-exchange chromatography (HPAEC) respectively were developed for detecting and characterizing the compositive and structural differences of polysaccharides extracted from the Ganoderma lucidum spore of different sources, and established two kinds of chromatographic fingerprints of the polysaccharides. Furthermore, we investigated the promoting effect of the polysaccharides from Ganoderma lucidum spore on lymphocyte proliferation by means of MTT experiments, which were applied to research the profile-effect relationship of immunocompetence.
To get better characterization of Ganoderma lucidum polysaccharides information through the fingerprints, the conditions of TFA partial hydrolysis were investigated and optimized. It was discussed that the effect about TFA concentration, hydrolysis temperature and hydrolysis time on the total peak area of the common peaks in the PMP-HPLC analysis. And the best TFA partial hydrolysis conditions were obtained: hydrolysis with 0.4 mol/L of TFA under 110℃for 70 min.
Following the TFA partial hydrolysis under the conditions above, the fingerprints of Ganoderma lucidum spore polysaccharides was established by use of the software Similarity Evaluation Eystem for Chromatographic Fingerprint of TCM (2004A), with 19 common peaks, the total peak area of which accounts for more than 95% of all peak area. The results of fingerprints similarity analysis of 15 samples of Ganoderma lucidum spore polysaccharides indicated that the similarity between products from the same origin was much higher than 0.97, while it was lower but still greater than 0.80 when the products originated in different areas. And significant differences could be found from the polysaccharides samples which are not from Ganoderma lucidum spore but under the same way of analysis.
Similarly, in order to get the better ion chromatographic fingerprints, gradient elution conditions were optimized. Under the optimal gradient elution conditions above, the conditions of acid-enzyme partial hydrolysis were also investigated and optimized. It was discussed that the effect about H2SO4 concentration, hydrolysis temperature and hydrolysis time on the two indicators-the total peak area of the common peaks of monosaccharides and oligosaccharides fractions respectively in the HPAEC analysis. And the best hydrolysis conditions were obtained: hydrolysis with 3 mol/L of H2SO4 under 90℃for 2 h before enzymolysis. Under these hydrolysis conditions, the fingerprints of monosaccharides and oligosaccharides of Ganoderma lucidum polysaccharides hydrolysates were established respectively.
In order to investigate profile-effect relationship of Ganoderma lucidum spore polysaccharides, MTT experiments were selected to reflect the immunocompetence of the polysaccharides. The proliferation data as the MTT experiments results of 20 samples of Ganoderma lucidum spore polysaccharides were got, with the results of HPAEC analysis of these samples, a multiple linear regression equation was established by using SPSS 17.0.
为了使指纹图谱更好地表征灵芝多糖的特征信息,本课题对灵芝多糖TFA部分水解的条件进行了考察。通过对TFA浓度、水解温度和水解时间进行单因素以及正交试验,以共有特征峰的峰面积和作为衡量指标,确定了最优水解条件:TFA浓度0.4 mol/L、水解温度110℃和水解时间70 min。
在上述水解条件下对15个灵芝孢子粉多糖样品进行PMP-HPLC分析,然后运用“中药色谱指纹图谱相似度评价系统2004A版”软件处理,建立了灵芝孢子粉多糖的PMP-HPLC标准指纹图谱,其中确定了19个共有特征峰,且共有峰峰面积和占总峰面积的95%以上。对于这15个样品指纹图谱相似度分析的结果表明,同一产地不同批次产品间的相似度很高,大于0.97;不同产地产品间的相似度较低,但均大于0.80。而对赤芝孢子粉以外的其它灵芝多糖类样品进行同样分析,则能够看到明显差异。
同样,为了得到理想的离子色谱指纹图谱,本课题对离子色谱分析条件进行了优化。在此条件下也对灵芝孢子粉多糖样品离子色谱分析前的酸-酶部分水解条件进行了考察。通过对硫酸浓度、水解温度和水解时间的单因素以及正交试验,综合考虑水解物中单糖和寡糖组分的共有特征峰峰面积和这两个指标后,确定了最优水解条件:硫酸浓度3 mol/L、水解温度90℃和水解时间2 h。在此条件下我们分别建立了灵芝孢子粉多糖水解产物单糖和寡糖组分的HPAEC标准指纹图谱。
为了考察灵芝孢子粉多糖的谱效关系,我们选择MTT实验表征灵芝孢子粉多糖的免疫活性。利用SPSS 17.0软件对20个样品MTT实验结果与其离子色谱指纹图谱中单、寡糖共有峰的相对峰面积数据进行多元回归分析,得到能够表征灵芝孢子粉多糖免疫活性的多元线性回归方程。
As one of the traditional fungi for both edible and medicinal use, Ganoderma lucidum has a history of more than two thousand years in China. With in-depth studies of components in Ganoderma lucidum such as polysaccharides, triterpenoids, alkaloids, adenosines and sterols, its physiological activities arrest more and more attention. Ganoderma lucidum polysaccharides, an outstanding representative of physiologically active substances among them, have played its important role as Ganoderma lucidum have come to market in the form of health care products, pharmaceuticals and functional food. Ganoderma lucidum and its products have mushroomed, while the quality of these products on the market is hard to distinguish, as well as to tell a true from a false. As the complexity of the composition and structure of polysaccharides, there is still a lack of analysis methods and quality standards which could effectively characterize and identify the polysaccharides from Ganoderma lucidum with physiological activity. In order to resolve the problem, we took the broken Ganoderma lucidum spore powder as the experimental material, and extracted Ganoderma lucidum polysaccharides by ultrasound-assisted extraction method. In our study, two methods of TFA partial acidic hydrolysis coupled PMP pre-column derivatization reverse phase high performance liquid chromatography (RP-HPLC) and acid-enzyme partial hydrolysis coupled high-performance anion-exchange chromatography (HPAEC) respectively were developed for detecting and characterizing the compositive and structural differences of polysaccharides extracted from the Ganoderma lucidum spore of different sources, and established two kinds of chromatographic fingerprints of the polysaccharides. Furthermore, we investigated the promoting effect of the polysaccharides from Ganoderma lucidum spore on lymphocyte proliferation by means of MTT experiments, which were applied to research the profile-effect relationship of immunocompetence.
To get better characterization of Ganoderma lucidum polysaccharides information through the fingerprints, the conditions of TFA partial hydrolysis were investigated and optimized. It was discussed that the effect about TFA concentration, hydrolysis temperature and hydrolysis time on the total peak area of the common peaks in the PMP-HPLC analysis. And the best TFA partial hydrolysis conditions were obtained: hydrolysis with 0.4 mol/L of TFA under 110℃for 70 min.
Following the TFA partial hydrolysis under the conditions above, the fingerprints of Ganoderma lucidum spore polysaccharides was established by use of the software Similarity Evaluation Eystem for Chromatographic Fingerprint of TCM (2004A), with 19 common peaks, the total peak area of which accounts for more than 95% of all peak area. The results of fingerprints similarity analysis of 15 samples of Ganoderma lucidum spore polysaccharides indicated that the similarity between products from the same origin was much higher than 0.97, while it was lower but still greater than 0.80 when the products originated in different areas. And significant differences could be found from the polysaccharides samples which are not from Ganoderma lucidum spore but under the same way of analysis.
Similarly, in order to get the better ion chromatographic fingerprints, gradient elution conditions were optimized. Under the optimal gradient elution conditions above, the conditions of acid-enzyme partial hydrolysis were also investigated and optimized. It was discussed that the effect about H2SO4 concentration, hydrolysis temperature and hydrolysis time on the two indicators-the total peak area of the common peaks of monosaccharides and oligosaccharides fractions respectively in the HPAEC analysis. And the best hydrolysis conditions were obtained: hydrolysis with 3 mol/L of H2SO4 under 90℃for 2 h before enzymolysis. Under these hydrolysis conditions, the fingerprints of monosaccharides and oligosaccharides of Ganoderma lucidum polysaccharides hydrolysates were established respectively.
In order to investigate profile-effect relationship of Ganoderma lucidum spore polysaccharides, MTT experiments were selected to reflect the immunocompetence of the polysaccharides. The proliferation data as the MTT experiments results of 20 samples of Ganoderma lucidum spore polysaccharides were got, with the results of HPAEC analysis of these samples, a multiple linear regression equation was established by using SPSS 17.0.
引文
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