Study on HPLC fingerprint and chemical pattern recognition of the Miao medicine Rolling Mountain Pearl
Prionobelum qianensis Chen is a dry whole insect of the genus Prionobelum in the Zephronidae family. It has a flattened cylindrical body and inhabits damp slopes, dead branches, leaves, or rocks. It is mainly distributed in Guizhou, Yunnan, and other regions. Rolling Mountain Pearl is a medicinal whole worm that has the effects of clearing heat, reducing swelling and pain, relaxing tendons and promoting blood circulation. It is mainly used to treat symptoms such as muscle spasms, bruises, fractures, swelling and pain. It is recorded in books such as “Chinese Materia Medica – Miao Medicine Volume” and “Guizhou Miao Medicine Research and Development”. In the Miao ethnic areas of Guizhou, the treatment for inflammation is very extensive, and the therapeutic effect on tumors is extremely significant. However, the current basic research on rolling mountain bead both domestically and internationally is very weak, and there is a lack of quality control standard system, which seriously restricts the rational development and widespread utilization of rolling mountain bead medicinal materials.
Fingerprint spectrum is a reliable means of identifying the authenticity, evaluating quality consistency, and stability of traditional Chinese medicine chemical components obtained through techniques such as spectroscopy or chromatography, based on the understanding of the overall action of substance groups. Therefore, in this study, HPLC was used to establish the fingerprint of Gongshanzhu, and similarity evaluation was conducted. Chemical pattern recognition methods such as cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to screen out the main characteristic components that cause differences in the composition of different batches of Gongshanzhu. The aim is to provide reference for the quality control and further research of Miao medicine Gongshanzhu.
In the early stage of the experiment, the influence of methanol and acetonitrile elution systems on the chromatographic peak separation efficiency was investigated. The results showed that methanol had better separation efficiency and a stable baseline when used as the mobile phase; We investigated the separation efficiency of methanol-0.1% formic acid water and methanol-0.1% acetic acid water, and ultimately selected the methanol-0.1% acetic acid water system with better separation efficiency; Analyzed the spectrum of full wavelength scanning at 210-240nm, showing that there are more peaks at 254nm, a stable baseline, and a large signal response value; By examining the effects of different flow rates (0.8, 1.0, 1.2 mL/min) and column temperatures (25, 30, 35, 40 ℃) on chromatographic peaks, it was determined to select a flow rate of 0.8 mL/min and a column temperature of 25 ℃. In the early stage of the experiment, two extraction methods, ultrasound and reflux, were also investigated, and it was found that there was no significant difference in their extraction efficiency. Therefore, ultrasound extraction, which has a simpler operation, was chosen; Comparing water, methanol, and edible alcohol as extraction solvents, the results showed that edible alcohol had more chromatographic peaks and larger response values during extraction. And the extraction effects of different concentrations of edible alcohol (30%, 50%, 70%, 90%) were compared. The results showed that the chromatographic peak response value was higher when 70% edible alcohol was extracted. Taking into account all factors, 70% edible alcohol ultrasonic extraction is selected as the preparation method for the test solution.
This study established for the first time the fingerprint spectrum of the Miao medicine Rolling Mountain Pearl, and the similarity of the fingerprint spectra of 24 batches of samples ranged from 0.739 to 0.957, identifying one component. Through chemical pattern recognition analysis, a classification model was established for the clustering analysis, principal component analysis, and orthogonal partial least squares discriminant analysis of rolling mountain pearls, clarifying the classification status between samples. 24 batches of samples were grouped into 3 categories, and 5 main characteristic components affecting the quality differences among the medicinal materials of Gongshanzhu were screened. In summary, the HPLC fingerprint method established in this study is stable, reliable, and has good reproducibility, which can provide a reference for the quality control of the medicinal herb Gongshanzhu.