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Complex Deng-Zhan-Sheng-Mai herbal mixture present analytical difficulties
The Chinese medicine Deng-Zhan-Sheng-Mai, used to treat stroke patients, consists of a mixture of four different herbal extracts, each containing a wide variety of compounds, thus giving a huge headache to analysts.
Deng-Zhan-Sheng-Mai is a Chinese medicine used to treat stroke patients. A trial reported in the Chinese Journal of Neurology in 2008 suggested that the mixture was effective in preventing strokes in high-risk patients. However, the trial was open rather than double blinded, which is a possible source of error.
Deng-Zhan-Sheng-Mai is made up of four herbal extracts: ‘Erigerontis Herba’ (EH) from Erigeron breviscapus (a type of fleabane), ‘Ginseng Radix et Rhizoma’ (GR) from ginseng roots and rhizomes, Schisandrae Chinensis Fructus (SCF) from the berries of Schisandra chinensis (the ‘five-flavour berry’) and Ophiopogonis Radix (OR) from the tubers of Ophiopogon japonicus (mondo grass).
Each of the four herbal extracts contains many compounds, making the development of reliable analytical methods for QC purposes extremely challenging. Current QC methods tend to focus on two of the major compounds from EH: scutellarin (a flavone) and 4,5-O-dicaffeoylquinic acid (a phenolic acid). Previous academic researchers have tended to rely on the analysis of a number of the predominant compounds present, without employing any systematic chemometric methods.
LC-MS and chemometrics used on Deng-Zhan-Sheng-Mai mixture
The Chinese researchers extracted Deng-Zhan-Sheng-Mai with aqueous methanol using ultrasound, which were found to be the optimum conditions. The resulting extracts were analysed by LC-MS. A UHPLC system was used, with an aqueous acetonitrile gradient (with 0.1% formic acid in the aqueous mobile phase). Mass spectrometry was carried out with a multistage mass spectrometer with an electrospray (ESI) source. For qualitative analysis, the mass spectrometer was operated either as single stage MS, tandem MS (MS2) or three stage MS (MS3). All the quantitative analysis was carried out using tandem MS (MS2). Data were recorded in both positive and negative ion modes.
A total of 55 compounds were identified or tentatively characterized. Definitive identification was possible in the case of many of the major compounds, such as scutellarin and ginsenoside Rg1 (from ginseng), where reference materials were available. Comparison with LC-MS from single herbs enabled the researchers to determine which compounds came from which plant, with 26 coming from EH, 12 from GR, ten from SCF and seven from OR. The main classes of compound present were phenolic acids, flavonoids, triterpenoid saponins and lignans. Eighteen compounds, including representatives of each class, were chosen as markers for quantification on the basis of their high abundance or known biological activity. A method for quantification was developed using these 18 compounds, and then successfully validated with regard to linearity, LOD (limit of detection), LOQ (limit of quantification), precision, repeatability and stability.
Ten different batches of Deng-Zhan-Sheng-Mai were examined, and the percentage of each class of compound (phenolic acids, flavonoids, saponins and lignans) present in each was determined. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were applied to the data. The results showed that simply focusing on two compounds, as in the current analytical method, was a poor method for evaluating batches. The antioxidant activity of each of the ten batches was measured and compared to their composition using bivariate correlation analysis (BCA). The total amount of the 18 compounds chosen as markers, the total flavonoids and the total phenolic acids were all significantly correlated with the antioxidant activity.
Chemometrics gives improved analysis of Deng-Zhan-Sheng-Mai
On the basis of the chemometric results and previous work on their biological activities, nine compounds were suggested as potential chemical markers for routine analysis of Deng-Zhan-Sheng-Mai. Thus an analysis of 55 compounds, impracticable for routine QC, was greatly simplified to an analysis of only nine compounds, giving a suitable method to determine the quality of Deng-Zhan-Sheng-Mai capsules. The authors suggest that their methodology could be applied to other herbal medicines.
Related Links
Journal of Separation Science, 2016, Early View paper. Jiang et al. Qualitative and quantitative analysis of multiple components for quality control of Deng-Zhan-Sheng-Mai capsules by ultra high performance liquid chromatography tandem mass spectrometry method coupled with chemometrics.
Chinese Journal of Neurology, 2008, 41, 195-200. Chen et al. The effects of Deng Zhan Sheng Mai capsule on secondary and tertiary prevention of ischemic stroke.
Source: Spectroscopy Now