基于偏最小二乘法的赤芝子实体三萜类化合物抗氧化谱效关系的研究

Spectrum-effect Relationship Between HPLC Fingerprints and Antioxidant Activity of Triterpenes in Ganoderma lucidum Fruiting Bodies Using Partial Least Squares Regression

测定了64株赤芝(Ganoderma lucidum)子实体醇提物清除二苯基苦基苯肼(DPPH)自由基的活性和三萜指纹图谱,采用遗传算法筛选其指纹图谱中的26个共有峰,并标定出其中15个特征峰,运用偏最小二乘回归分析方法(partial least squares regression,PLSR)对指纹图谱中26个三萜峰与醇提物清除DPPH自由基的活性进行关联分析,建立了赤芝子实体三萜类化合物的抗氧化谱效关系方程。在赤芝子实体醇提物三萜指纹图谱的26个共有峰中,峰S1、S4、S9、S10和S26的峰面积与醇提物清除DPPH自由基的活性呈显著正相关关系;峰S3、S11、S15、S18、S19、S22和S25的峰面积与醇提物清除DPPH自由基的活性呈显著负相关关系。分别测定鉴定出的15个峰对应的三萜化合物对DPPH自由基的清除活性,发现与清除DPPH自由基活性呈显著正相关的三萜化合物灵芝酸C2(S4)、灵芝酸B(S9)、灵芝酸LM2(S10)、灵芝醛A(S26)对DPPH自由基的清除活性明显高于其他三萜化合物,与清除DPPH自由基活性呈显著负相关的三萜化合物灵芝酮三醇(S18)、灵芝萜酮二醇(S22)无清除DPPH自由基的活性。另外,利用H2O2诱导的PC12细胞损伤模型进一步验证灵芝酸C2、灵芝酸B、灵芝酸LM2、灵芝醛A和灵芝萜酮二醇在细胞水平的抗氧化活性,发现灵芝酸C2、灵芝酸B、灵芝酸LM2、灵芝醛A具有明显的保护神经细胞氧化损伤的活性,而灵芝萜酮二醇对神经细胞的氧化损伤无保护作用。结果表明,本研究建立的赤芝子实体三萜类化合物抗氧化谱效关系方程具有较高的准确性,能够较全面地反应赤芝子实体醇提物中不同三萜化合物与其抗氧化活性的相关性,可快速锁定有效成分以指导活性成分的分离纯化工作。此外,该方法也为赤芝子实体的质量控制提供了更为有效和准确的方法。

Fruiting bodies of 64 strains of Ganoderma lucidum were extracted by ethanol to obtain their ethanol extracts. These extracts were then determined for their 1,1-diphenyl-2-picrylhydrazyl(DPPH) scavenging rate and analyzed for their triterpenoid fingerprints. Using the genetic algorithm, 26 common peaks were screened and 15 characteristic peaks were demarcated. Then partial least squares regression(PLSR) was applied to set up a regression equation between DPPH scavenging activity of ethanol extracts and relative peak area of 26 triterpenes in the fingerprint. Among the 26 common peaks, peaks S1, S4, S9, S10, S26 were significantly positively correlated with DPPH scavenging activity, and peaks S3, S11, S15, S18, S19, S22 and S25 were significantly negatively correlated with DPPH scavenging activity. For the 15 characteristic triterpenes, ganoderic acid C2(S4), ganoderic acid B(S9), ganoderic acid LM2(S10), ganoderal A(S26) showed significantly higher DPPH scavenging activity than other positively correlated triterpenoids, whereas ganodermanontriol(S18) and ganodermanondiol(S22) had no DPPH scavenging activity. In addition, ganoderic acid C2, ganoderic acid B, ganoderic acid LM2 and ganoderal A showed both antioxidant and nerve cell protection activity against H2O2 induced oxidative damage on PC12 cells, while ganodermanondiol only showed antioxidant but no nerve cell protection activity. The spectrum-antioxidant effect equation established in this study has a high accuracy on reflecting antioxidant activities of different triterpenes in G. lucidum extracts. It can be used to guide separation and purification work by quick identification of an active ingredient. This method also provides an effective approach for quality control on G. lucidum fruiting bodies.