HPLC method development for characterisation of the phenolic composition of Cyclopia subternata and C. maculata extracts and chromatographic fingerprint analysis for quality control

[摘要] ENGLISH ABSTRACT: The phenolic composition of Cyclopia species is believed to be partially responsible for the numerous healthpromoting properties associated with their extracts. Current quality control measures do not accommodatevariation in phenolic profiles of Cyclopia species. In this study, comprehensive high performance liquid chromatography(HPLC) methods were developed for the improved characterisation of the phenolic composition ofaqueous extracts of two Cyclopia species (C. subternata and C. maculata). The methods were developed to besuitable for both routine quantitative analysis on conventional HPLC instrumentation, and the construction ofchromatographic fingerprints for further data analysis. The latter entailed similarity analysis and prediction oftotal antioxidant capacity (TAC).Using a methodical approach, two separate HPLC methods, using diode array detection (DAD), were developedand validated for the analysis of aqueous extracts prepared from unfermented (green) and fermentedplant material of C. subternata and C. maculata. Separation was achieved using the same method parameters(column, temperature, mobile phases), except for differing mobile phase gradients. Hyphenation ofthe developed HPLC methods with mass spectrometry (MS) and tandem MS allowed the confirmation ofphenolic compounds previously identified in Cyclopia, and the tentative identification of several additionalcompounds in Cyclopia species, which are reported here for the first time. These included apigenin-6,8-di-C-glucoside, 3-hydroxyphloretin-30,50-di-C-hexoside, eriodictyol-di-C-glucoside, iriflophenone-di-O,C-hexoside,hydroxymangiferin and hydroxyisomangiferin. Subsequently, a large number of aqueous extracts of randomlyselected green C. subternata (n = 64) and C. maculata (n = 50) plant material samples were analysed. Largequantitative variations were observed on intra- and inter-species levels. Cyclopia maculata extracts containedalmost six times more mangiferin than extracts from C. subternata.HPLC-DAD analysis produced duplicate fingerprints for each extract which were consequently used forfurther analysis. The chromatographic fingerprint of a bioactive extract of each species was included in therespective data sets. Similarity analysis was conducted between the fingerprints from the randomly selectedextracts and the corresponding active extract. For each species several extracts were determined to have similar'activity as that of the active extract (n = 15 for C. subternata and n = 45 for C. maculata). Compoundspotentially responsible for the activity were tentatively identified with the aid of principal component analysis(PCA) in combination with similarity analysis. PCA was more effective in identifying small differences betweenfingerprints than similarity analysis based on the correlation coefficients (r) alone.Furthermore, multivariate data analysis was used to construct partial least squares (PLS) regression modelsfor the prediction of TAC from fingerprint data of each species, and available data from two microplate TACassays. The construction of the models was successful with reasonable errors (< 10%), and permitted thedetermination of compounds of interest for future research. These included compounds of known identity thathad large positive contributions toward the predictions of TAC, or unknown compounds that had small UV signals, but relatively large positive contributions to the models.

[发布机构] Stellenbosch University