Chemometrics Assisted Gas Chromatography-Mass Spectrometry For Determination Of Flos Magnoliae Volatile Oil And Its Anti-inflammatory Mechanism Research

Based on the problems faced by complex system of Chinese medicine qualitative and quantitative analysis and the research of pharmacodynamics mechanism, in the present paper, Flos magnoliae volatile oil was chosen as the research subject, and gas chromatography-mass spectrometry was used as analytical tools and chemometric resolution methods and metabonomics research techniques were carried out to conduct a comprehensive and systematic study. This research provides the basis for pharmacological activity of Flos Magnoliae volatile oil, and gives a certain amount of reference and help for pharmacodynamic study of Chinese medicine formulation containing Flos Magnoliae.Some studies are as follows,1. Retention time prediction of target chromatographic peaks in gas chromatography conditionWhen one non-isothermal run and another isothermal run were used to predict the retention time in capillary gas chromatography, the relative error is large; when two one-step programmed runs were used, the relative error is small. In the case, the relatively larger error could be due to the relatively larger difference of the sample in partition between under the non-isothermal condition and under the isothermal condition.Based on real needs, a modified method was proposed for retention time prediction in two-step temperature-programmed gas chromatography, based on the closed-form integral method which was formerly put forward to predict the retention time in one-step gas chromatography. The calculation process was simplified and the prediction error was reduced in the proposed method. The modified method was validated through predicting the retention time of two reference standards, n-hexadecanoic acid and nonacosane using two-step temperature-programmed gas chromatography, and the relative errors of prediction were less than 0.2%. Then the proposed method was applied to predict the retention time of Flos Magnoliae volatile oil, and satisfactory results were obtained as well. The relative errors of prediction were less than 0.15%. The results indicate that our modified method is effective for retention time prediction in the two-step temperature-programmed gas chromatography, and the prediction error could be reduced using the proposed method. The prediction method can be used to optimize the gas chromatographic conditions, save analysis time and improve the analysis efficiency. Thus, there are good application field in practical work.2. Determination of the volatile oil of Magnolia biondii Pamp by GC-MS combined with Chemometric TechniquesAn optimized temperature-programmed gas chromatography-mass spectrometry system combined with chemometric methods was firstly applied to analyze the volatile components of M. biondii Pamp (a kind of Flos Magnoliae). The peak purity of two-way data was controlled by fixed size moving window evolving factor analysis, two dimensional-evolving latent projection graph and three dimensional-evolving latent projection graph. Then the overlapped peak clusters were resolved using heuristic evolving latent projection. We choose peak cluster A and B as examples to illustrate the data analysis process. The results show that the components co-eluted in peak cluster A is o-cymene, D-limonene and eucalyptol. Their relative contents are 2.27%, 4.52% and 14.20%, respectively; the components co-eluted in peak cluster B is Carotol,τ-Cadinol,τ-Muurolol and 1á-Cadin-4-en-10-ol. Their relative contents are 0.13%, 1.41%, 1.01% and 0.41%, respectively. A total of 65 components were identified using similarity searches between mass spectra and MS database. This number was extended to 80 components with the help of chemometric techniques.In this study, we make full use of the 3D-ELPG information, and more accurate and reliable analytical results can be acquired. The research indicates GC-MS combined with chemometric techniques can be successfully used to analyze the volatile oil of Magnolia biondii Pamp, and the CRM method in analysis of complex systems have the potential advantages. The accuracy of qualitative and quantitative analysis can be improved. The results also prove that the reported approach is powerful for the analysis of complex herbal samples. 3. Comparative study on fingerprints of Flos Magnoliae volatile oil by GC-MS combined with MCR-ALSTwelve different sources of Flos Magnoliae were analyzed and compared with each other. The overlapped chromatographic peaks of 12 batches of Flos Magnoliae volatile oils were resolved through MCR-ALS method, and the accuracy of qualitative and quantitative was increased. It is the first time to apply MCR-ALS method to resolve the chromatographic peaks in fingerprint research, and it reduces the burden of qualitative analysis as well as the subjectivity. Among the components were determined, there were 34 components coexisting in all samples. Eucalyptol, (E, E)-Farnesol,α-phellandrene, Camphene, D-Limonene,α-Cadinol,α-Linalool, Caryophyllene and o-cymene are the main components in Flos Magnoliae volatile oil. N strong peaks, Common and Variant peak ratios were used to evaluate their fingerprint. The results showed a fair consistency in their GC-MS fingerprint.4. Quantitative analysis and preliminary study on pharmacokinetics of 1, 8-cineole in rats plasmaA gas chromatography method has been developed for the determination of 1,8-cineole in rat plasma usingα-pinene as internal standard. Liquid-liquid extraction was used as the sample pretreatment mode. Separation was obtained on a 30 m×0.25 mm i.d. capillary column coated with 0.25μm film SPBTM-1. Nitrogen was used as carrier gas at a 1.0 mL/min flow rate. To acquire good separation, the column temperature was initially maintained at 50°C for 2 min, and then increased from 50 to 100°C at a rate of 5°C/min, and held for 2 min. The chromatographic system used in this research provided good separation of compounds without interfering peaks from endogenous substances. The calibration curve was liner range with correction coefficients above 0.999. The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 3.75~5.74% and 2.41~5.58%, respectively. The method has been successfully used to support the pharmacokinetics study of 1,8-cineole.Following a single oral administration with 400mg/kg to rats, the blood samples were collected at different time after dosing. All collected blood samples were centrifuged to obtain plasma and the concentration of 1,8-cineole in plasma were determined by gas chromatography method described as above. Pharmacokinetics parameter calculations were carried out using non-compartmental analysis method. It showed from the plasma concentration-time data that the concentrations of 1,8-cineole in rat plasma increased and then reduced slowly after oral administration and the individual difference of rats was evident. The peak plasma concentration was 36.223μg/ml at 2h. The estimated elimination half-time and Mean residence time in rats were 4.811h and 7.923h, respectively. AUC0~12 and AUC0~∞were 227.09μg·h/ml and 280.06μg·h/ml, respectively.These results indicated that 1,8-cineole has slow oral absorption and its biological half-time period is relative longer. If suitable pharmaceutical dosage forms could be used, its bioavailability would be higher. In addition, the relevant research suggests that 1,8-cineole has a strong permeability, it can be developed for the transdermal formulations as local anti-inflammatory or antibacterial drug.5. Intervention effect of Volatile Oil of Magnolia biondii Pamp (VOMbP) on rat model of acute inflammation: A plasma metabonomics study based on gas chromatography-mass spectrometryIn the present study, GC-MS-based metabonomics was applied to investigate the metabolic profiles of rats with acute inflammation induced by carrageenan, and the intervention effects of VOMbP on acute inflammation were evaluated as well. The endogenous metabolites in rat plasma were identified by NIST library, and the metabolic patterns were investigated using principal component analysis. The PCA scores plot showed that the inflammation group was apparently separated to the control group, indicating that the two groups had completely different metabolic profiles. When PCA was applied to treat the data of the control, inflammation and VOMbP groups, however, it can be seen that the three groups were apparently apart from each other, whereas the VOMbP group and the control group were adjacent at the same side on PC2. This indicated that the metabolic profile of VOMbP pretreated group was totally different from that of the model group and was showing a trend to return to the control group.In the corresponding loadings plot based on the GC-MS data of three groups, the metabolites which are the furthest from zero could be considered as marker metabolites that contribute most strongly to the classification of the three groups. In inflammation group, levels of urea, cadaverine, glycine and lactic acid increased, whereas the other metabolites had reduced levels. After VOMbP pretreatment, however, it can be seen that the levels of urea, cadaverine, glycine and lactic acid were reduced and the levels of the other metabolites were elevated comparing to those in the inflammation group. Among them, six metabolites, namely hexadecanoic acid, linoleic acid, oleic acid, stearic acid, arachidonic acid and cholesterol, may be mostly related to the anti-inflammatory activity of VOMbP may be involved in the anti-inflammatory activation of VOMbP, these metabolites may be regarded to act as LXRs activators to hinder the release of the inflammatory mediators and inhibit the inflammatory response. Therefore, VOMbP may exert its effectiveness on anti-inflammatory activation by involving the LXRs signaling pathway.