Identification of UPLC characteristic spectra of four original plants of Xihuangcao medicinal herbs
Xihuang Grass is a unique Chinese herbal medicine in the south of the Five Ridges, which has the effect of clearing heat and removing dampness, cooling blood and removing stasis. It is used for damp heat jaundice, abdominal distention and hypochondriac pain, damp heat diarrhea, heat toxin diarrhea, and traumatic injuries. It is a commonly used clinical medicine in the south of the Five Ridges, and is also a raw material for the prescription of the famous traditional Chinese patent medicines and simple preparations big variety “anti-inflammatory and cholagogue tablets”. The Chinese Pharmacopoeia (2020 edition, Volume 4) records it as the dried aboveground part of Rabdosia lophanthoides (Buch. – Ham. ex D. DON) H. Hara or Rabdosia serora (Maxim.) H. Hrar, a plant belonging to the Labiatae family and the genus Rabdosia. The original plant included in the Guangdong Provincial Chinese Herbal Medicine Standards is Rabdosia lophanthoides R serra、 Thread patterned fragrant tea and vegetable R Lophanthoides and its fibrous flower variety R lophanthoides var. graciliflora(Benth.) H. Hrar。 During the process of researching and organizing the germplasm resources of Xihuangcao medicinal materials, members of our research group found that Rabdosia stracheyi (Benth. ex Hook. f.) Hara, a plant belonging to the same genus, is also commonly used as Xihuangcao medicinal materials in folk culture. Due to the similar morphological characteristics of these plants, it is difficult to distinguish dry medicinal materials based on their appearance, and there has always been a phenomenon of confusion in the variety resources of Xihuangcao commercial medicinal materials. Therefore, it is of great significance to establish identification methods for different types of Huangcao in order to ensure and improve its clinical drug safety and efficacy.

Traditionally, the identification of different original plants of Xihuangcao medicinal materials has been studied from the aspects of morphology and microscopy. However, due to the fact that dry medicinal materials are often wrinkled and broken, it is difficult to accurately and quickly identify them. In recent years, scholars have conducted identification studies on its original plants at the level of DNA molecules and chemical components. However, molecular identification can only distinguish between Rabdosia serora and Lycaena angustifolia at the species level, making it difficult to distinguish between the original and variant varieties of Lycaena angustifolia. In terms of chemical composition research, some scholars believe that Fourier transform infrared spectroscopy and two-dimensional correlation infrared spectroscopy can achieve the identification of Xihuangcao, Lined Fragrant Tea Vegetable and its fibrous flowers and narrow base varieties. Some scholars have also conducted research on the identification of chemical composition content and fingerprint spectra, but the experimental results cannot distinguish the original variety of Lined Fragrant Tea Vegetable from its variants very well. In recent years, chemometric methods have been increasingly widely used in the chromatographic analysis of traditional Chinese medicine. They can further analyze and process the large amount of data generated by chromatographic analysis, and play an increasingly important role in the quality control of traditional Chinese medicine when combined with chromatographic analysis. Ultra high performance liquid chromatography (UPLC) has the advantages of short analysis time, good separation effect, and high sensitivity. Therefore, this study used UPLC to study the characteristic spectra of 32 batches of collected medicinal materials from four different base plants, namely Xihuangcao, Xianwen Xiangchacai, Xianxiang Xiangchacai, and Longye Xiangchacai, and combined with chemometric methods such as cluster analysis, principal component analysis, and partial least squares discriminant analysis, to distinguish and identify these four base plants, in order to provide valuable insights for Xixi. Provide reference for the identification of Huangcao medicinal materials and the analysis and evaluation of their quality.

 

This study screened and optimized the preparation methods and chromatographic conditions of the test solution. The investigation results of extracting solvents (methanol, 80% methanol, 60% methanol) showed that when methanol was used as the extraction solvent, the obtained sample chromatogram had more chromatographic peaks, relatively larger peak areas, and a stable baseline, which better reflected the chemical composition of Xihuangcao medicinal material. Therefore, methanol was selected as the extraction solvent for the characteristic spectrum of Xihuangcao medicinal material; The results of measuring wavelengths (250, 254, 260, 280nm) showed that there were many chromatographic peaks at 254nm, and the sizes of each peak were uniform. Therefore, 254nm was selected as the detection wavelength; The investigation results of mobile phases (methanol -0.1% formic acid water, acetonitrile -0.1% formic acid water, 0.1% formic acid methanol -0.1% formic acid water, and 0.1% formic acid acetonitrile -0.1% formic acid water) showed that when using methanol -0.1% formic acid water as the mobile phase for elution, the baseline was flat, and the separation degree and peak shape were good; The results of column temperature and flow rate investigation showed that the separation efficiency of each chromatographic method was the best when the column temperature was 30 ℃ and the flow rate was 0.3mL/min.

In this study, UPLC was used to establish a characteristic map applicable to the identification of four basic plants of Xihuangcao medicinal herb. The baseline was flat, the separation degree was good, and the number of common peaks was large. In the established control maps of Xihuangcao, Xianwen Xiangchacai, Xianxiang Xiangchacai, and Longye Xiangchacai, 19, 24, 26, and 21 common peaks were calibrated respectively. Based on the differences in their common peaks, the four basic plants can be preliminarily distinguished. Due to the small number of collected batches of Xianwen Xiangchacai and Longye Xiangchacai, in order to avoid errors in the research process, chromatographic peaks with higher response values, better separation degree, and peak shape were selected for calibration when studying common peaks. The similarity evaluation results indicate that due to differences in place of origin, there are certain differences between the same original medicinal materials; There are also differences between different base medicinal materials, among which the similarity between Xihuangcao and the other three base medicinal materials is relatively low and can be clearly distinguished; The similarity between Xianhua fragrant tea vegetable and Xianwen fragrant tea vegetable is relatively high, and the similarity between Changye fragrant tea vegetable and Xianhua fragrant tea vegetable is also relatively high. The reason may be that Xianhua fragrant tea vegetable, Xianwen fragrant tea vegetable, and Changye fragrant tea vegetable are all plants belonging to the wrinkled leaf fragrant tea vegetable group, with similar chemical composition types. Existing research has shown that there is little difference in the types of chemical components contained in the original plants of Xihuangcao medicinal materials, which mainly include terpenes, flavonoids, phenolic acids, and sterols. Among them, diterpenes are important components of the medicinal herb Xihuangcao. The main type of diterpenes contained in the original plant Xihuangcao is the enantiomeric kaempferol type, while the linear fragrant tea vegetable and its fibrous flower variety, as well as the long leaf fragrant tea vegetable, mainly contain the rosin type and enantiomeric rosin type diterpenes. It is speculated that the low similarity between Xihuangcao and linear fragrant tea vegetable, fibrous fragrant tea vegetable, and long leaf fragrant tea vegetable may be related to the differences in the types of diterpenes, while the differences between linear fragrant tea vegetable, fibrous fragrant tea vegetable, and long leaf fragrant tea vegetable may be related to the content and types of their respective components.
Principal component analysis showed that Xihuangcao can be further distinguished from Lined Fragrant Tea Vegetable, Fragrant Tea Vegetable, and Long leaved Fragrant Tea Vegetable by the difference in principal component scores of each base element. Partial least squares discriminant analysis can distinguish three types of primitive plants, namely line patterned fragrant tea plant, fibrous fragrant tea plant, and long leaved fragrant tea plant, with significant classification effects. The 15 classification markers obtained are the main characteristic components that cause differences among the three, which are of great significance for distinguishing these three different primitive plants. The identification of these 15 chromatographic peaks still needs further research and identification.
In summary, the UPLC characteristic maps of the four basal plants of Xihuangcao established in this study can preliminarily distinguish the four different basal plants through the calibration of characteristic peaks and similarity evaluation. In order to better distinguish the four types of original plants and search for differential components, this study further used cluster analysis, principal component analysis, and partial least squares discriminant analysis to completely distinguish the four types of original plants in Xihuangcao. The research results provide a scientific method and reference for the identification, quality analysis, and evaluation of the original plants in Xihuangcao medicinal materials.