Research on chrysanthemum characteristic markers based on HPLC characteristic spectra combined with chemometrics
Chrysanthemum is the dried head inflorescence of the Asteraceae plant Chrysanthemum morifolium Ramat. It mainly contains active parts such as flavonoids, volatile oils, organic acids, and trace elements. It has the effects of dispersing wind, clearing heat, calming the liver, improving vision, and clearing heat and detoxifying. The 2020 edition of the Chinese Pharmacopoeia includes chrysanthemum varieties such as “Boju”, “Chuju”, “Gongju”, “Hangju”, and “Huaiju” according to their place of origin and processing methods. As a Chinese medicinal herb with medicinal and edible properties, chrysanthemum has the characteristics of a wide variety and complex composition of ingredients. However, different varieties of chrysanthemum have significant differences in chemical composition due to their different growth environments, cultivation and processing methods, and uneven quality, resulting in different clinical therapeutic effects. At present, the 2020 edition of the Chinese Pharmacopoeia uses the content of three chemical components, chlorogenic acid, luteolin-7-O – β – D-glucoside, and 3,5-O-dicaffeoylquinic acid, as evaluation indicators, which makes it difficult to comprehensively evaluate the quality of chrysanthemum medicinal materials.
The characteristic spectrum of traditional Chinese medicine can characterize the overall changes in the internal quality of traditional Chinese medicine. More and more traditional Chinese medicines use the establishment of characteristic fingerprint spectra for quality control, among which HPLC characteristic fingerprint technology is also commonly used in the identification of chrysanthemum varieties and quality control research. The traditional Chinese medicine quality marker (Q-Marker) is composed of exclusive, clinically effective, and monitored chemical components that are related to the functional properties of traditional Chinese medicine. It can reflect the safety and effectiveness of traditional Chinese medicine and can be used to construct a modern comprehensive, systematic, and quantitative quality evaluation system for traditional Chinese medicine. On the basis of Q-Marker, by integrating fingerprint spectra and chemometric methods, components that reflect common characteristics in medicinal herbs or formulas can be discovered, and a multidimensional quality evaluation system that conforms to the characteristics of traditional Chinese medicine can be established. This may provide a better solution for the scientific supervision of traditional Chinese medicine.
On the basis of previous research on chrysanthemums, this study established HPLC characteristic chromatograms of different varieties of chrysanthemums, combined with chemometric methods to screen the common substance basis of chrysanthemums as characteristic markers. At the same time, significant differences in characteristic markers between different varieties of chrysanthemums were screened, providing scientific basis for overall quality evaluation, variety identification, and single variety characteristic evaluation of chrysanthemums.

 

Selection of extraction methods. This experiment investigated the effects of different solvents (70% methanol, 50% methanol, 80% methanol), extraction methods (ultrasonic extraction and heating reflux extraction), and extraction times (30, 35, 40 minutes) on chromatograms, and determined to use 70% methanol ultrasonic extraction for 40 minutes as the extraction method.
Optimization of chromatographic conditions. This experiment examined different chromatographic columns (Agilent XDB-C18 column Phenomenex Luna C18)、 Detection wavelengths (220, 254, 330, 348nm) and mobile phase systems (acetonitrile -0.1% phosphoric acid water, acetonitrile -0.1% formic acid water, acetonitrile -0.2% acetic acid water) were used, and the chromatographic peak shape, number of peaks, and resolution were used as evaluation indicators. Finally, an Agilent XDB-C18 chromatographic column (4.6mm × 250mm, 5 µ m) was selected with a detection wavelength of 348nm and acetonitrile -0.2% acetic acid aqueous solution as the mobile phase system.
The five different varieties of chrysanthemum control maps established in the experiment have different common peaks and identified characteristic peaks. In the established control maps of Boju, Chuju, Gongju, Hangju, and Huaiju, 21, 20, 25, 22, and 22 common peaks were respectively identified, and 14, 14, 12, 13, and 13 main characteristic peaks were identified by comparing with the control sample; In the chrysanthemum control spectrum generated from 50 batches of chrysanthemum samples, 17 common peaks were identified, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, luteolin-7-O – β – D-glucoside, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, apigenin 7-O – β – D-glucoside, isochlorogenic acid C, vanillolignin 7-O – β – D-glucoside, vanillogenin, apigenin, and luteolin. As caffeic acid was not identified in Gongju, Hangju, and Huaiju, and acacetin was not identified in Gongju, caffeic acid and acacetin were not used as common peaks in the chrysanthemum control spectrum. By comparing the peak areas, it was found that there were significant differences in the peak areas corresponding to luteolin-7-O – β – D-glucoside, apigenin, apigenin 7-O – β – D-glucoside, and isochlorogenic acid C among the five chrysanthemum control spectra. In the Gongju and Hangju control spectra, the peak areas of luteolin-7-O – β – D-glucoside and isochlorogenic acid C were larger, while in the other three chrysanthemum control spectra, their peak areas were smaller; In the control spectra of Boju, Chuju, and Huaiju, the peak area of apigenin is relatively large, while in the control spectra of Gongju and Hangju, its peak area is very small; The peak area of apigenin 7-O – β – D-glucoside is larger in the Hangzhou chrysanthemum control spectrum, while it is smaller in the other four chrysanthemum control spectra. So by comparing the differences between the characteristic peaks of the five varieties of chrysanthemums, we can preliminarily reflect the differences between different varieties of chrysanthemums, indicating that there are certain differences in the types and contents of chemical components in different varieties of chrysanthemums.
The similarity evaluation results show a high intra species similarity, indicating that the differences between chrysanthemums of the same variety from different batches are relatively small; The low inter species similarity is 0.391-0.690, indicating significant differences in the chemical composition of different varieties of chrysanthemums. CA, PCA, and OPLS-DA can accurately classify five different varieties of chrysanthemums into five categories. Although the five varieties of chrysanthemums are different, they also have similarities. PCA and OPLS-DA divide the five specific regions into two categories: left and right. Among them, Boju, Chuju, and Huaiju are classified as one category (for medicinal purposes), while Gongju and Hangju are classified as one category (for medicinal purposes). Chemometric analysis shows that coumarin-7-O – β – D-glucoside, isochlorogenic acid C, and apigenin are the main differential components of the two categories, which can be used as characteristic markers to distinguish the differences between the two types of chrysanthemums. OPLS-DA screened out 7 main marker components, and identified 4 components including apigenin, apigenin 7-O – β – D-glucoside, luteolin-7-O – β – D-glucoside, and isochlorogenic acid by comparing with mixed reference standards. These components can be used as characteristic markers to distinguish differences between different varieties of chrysanthemums, which are supported by the comparison of peak areas. Among them, peaks 24, 11, and 20 were not identified, and their roles in chrysanthemum quality evaluation need further research; Ten components with VIP values less than 1, including isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, vanillin, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, luteolin, and vanillin 7-O – β – D-glucoside, can be used as characteristic markers of chrysanthemum as a whole.
In summary, this experiment established and analyzed HPLC characteristic spectra of five chrysanthemum varieties, compared characteristic peaks and their corresponding peak areas, and conducted similarity evaluation, preliminarily demonstrating the differences between different chrysanthemum varieties; By using chemometric methods to analyze and screen the same substances among different varieties of chrysanthemums as characteristic markers representing chrysanthemum efficacy and reflecting the differences between different chrysanthemums, scientific basis is provided for chrysanthemum variety identification and quality evaluation.