Research on Quality Evaluation of Baimao Root Standard Soup from Different Origins Based on UPLC Fingerprint and Multi index Component Quantification
Selection of content indicators. The standard decoction of traditional Chinese medicine is a single herb decoction prepared by using Chinese herbal medicine slices as raw materials, referring to the traditional decoction habits of Chinese herbal medicine, using water as the solvent, and adopting a standardized decoction mode. As a standard substance or system, traditional Chinese medicine standard decoction is commonly used to standardize the clinical use of traditional Chinese medicine, standardize the use of new traditional Chinese medicine decoction pieces such as formula granules, and ensure the accuracy of clinical use of traditional Chinese medicine. Therefore, establishing quality standards for traditional Chinese medicine standard decoctions can provide important references for the establishment of quality standards for modern Chinese medicine formulations such as corresponding Chinese medicine formula granules and classic formulas.
The root of the Poaceae plant Imperata cylindrica Beauv The dried rhizome of var. major (Nees) C. E. Hubb. has the effects of cooling blood, stopping bleeding, clearing heat, and diuresis. Modern research has shown that the roots of Houttuynia cordata contain various chemical components such as triterpenoids, flavonoids, organic acids, phenylpropanoids, endosulfans, and polyols, which have various pharmacological activities such as hemostasis, diuresis, analgesia, antibacterial, anti-inflammatory, antioxidant, blood pressure lowering, liver protection, and regulation of immune function. The 2020 edition of the Chinese Pharmacopoeia lacks content indicators for the quality standards of Houttuynia cordata root medicinal materials, and the current quality standards are difficult to fully reflect the intrinsic quality of Houttuynia cordata root. Previous research reports have mostly used fingerprint/characteristic spectrum combined with chlorogenic acid content determination for quality control of Polygonatum sibiricum roots, but there has been no quantitative study on the combination of fingerprint spectrum and multiple indicator components. Research has shown that phenolic acid components in traditional Chinese medicine have anti-inflammatory, antibacterial, antioxidant, anti-tumor and other effects, and are natural active products with great research value and application potential. The root of Houttuynia cordata contains a large amount of phenolic acid components, and its pharmacological activities such as anti-inflammatory, antibacterial, and antioxidant are closely related to the functional indications of Houttuynia cordata root. Therefore, this study established the UPLC fingerprint of Houttuynia cordata root standard decoction and achieved simultaneous determination of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, and p-light cinnamic acid content in the standard decoction for the first time. Combined with chemometric analysis methods, it provides a basis for the quality evaluation of Houttuynia cordata root standard decoction in different production areas.

Selection of content indicators. At present, there is relatively little research on the indicators for determining the content of Polygonatum sibiricum roots, mainly focusing on the determination of phenolic acids and sugars. There are also reports on the determination of the content of Polygonatum sibiricum and Caryophyllin in Polygonatum sibiricum roots. However, the content of Polygonatum sibiricum and Caryophyllin in medicinal materials is very low. The standard decoction of Polygonatum sibiricum roots uses water as the solvent, and the active ingredients are mainly water-soluble components. Polygonatum sibiricum and Caryophyllin are not only low in content but also fat soluble components, so they are not suitable as indicator components. The specificity of sugar components is poor, and they are also not suitable as indicator components. From the study of fingerprint spectra, it was also found that although the roots of Polygonatum sibiricum have many chemical components, the content of effective ingredients is relatively low. Identify two chromatographic peaks with higher peak types from the fingerprint spectrum, both of which are phenolic acid components and closely related to the pharmacological activity of the standard decoction of Houttuynia cordata. Therefore, in the selection of content indicators, the determination of phenolic acid components is preferred. Research has shown that chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid are relatively stable under acidic pH conditions, but unstable under neutral and alkaline conditions, with a certain degree of conversion between them. In the process of studying the fingerprint spectra of Houttuynia cordata root and standard decoction, we found that compared to Houttuynia cordata root, the fingerprint spectra of Houttuynia cordata root standard decoction showed a significant increase in the peak area ratio of peak 1 (neochlorogenic acid), peak 8 (cryptochlorogenic acid), and peak 5. Therefore, we speculate that during the decoction process of Houttuynia cordata root, there is a higher probability of conversion of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid. Chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid have similar structures and pharmacological activities. Therefore, we believe that when selecting content indicators for the standard decoction of Zhimao root, it is best to determine the total amount of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid, rather than just measuring individual chlorogenic acid components. The research results show that there are significant differences in the total amount of four phenolic acid components in the standard decoction of Polygonatum sibiricum from different production areas, reflecting the quality of Polygonatum sibiricum medicinal materials from different production areas, which may be related to the different harvesting times and processing methods of medicinal materials from different production areas.

Establishment of analytical methods. At present, there is no research on the quality control of Houttuynia cordata root using fingerprint spectrum combined with multi-component quantification. The freeze-dried powder of Houttuynia cordata root standard decoction contains a lot of sugars, and the structure of phenolic acid components is similar, which requires high chromatographic separation. Therefore, in the establishment of the method, the separation effect of different mobile phase ratios, chromatographic columns, column temperatures, and flow rates on each fingerprint peak, especially the chromatographic peak for content determination, should be fully examined, and the optimal chromatographic conditions and columns should be selected; Select 280, 300, 325, and 350nm as the detection wavelengths and perform full wave scanning to record the absorption spectra of the sample in the range of 200-400nm. The results show that there is no significant difference in the number and peak types of each chromatogram at different wavelengths. Using 325nm as the detection wavelength, the absorption value of the chromatogram peak is the highest. Therefore, 325nm is selected as the detection wavelength for this characteristic spectrum. Using single factor analysis method, the extraction solvents (methanol, 10% methanol, 50% methanol, 70% methanol, 70% ethanol, 50% ethanol, 10% ethanol), extraction methods (ultrasound and heating reflux), solvent dosage (15, 25, 50mL) and extraction time (15, 30, 45min) were investigated to determine a reasonable preparation method for the test solution.

Similarity evaluation and chemometric analysis. The similarity calculation results of fingerprint spectra show that the similarity values of three batches of standard decoction of Polygonatum sibiricum in Henan production area are significantly lower than those in other production areas, indicating that there are significant differences in the chemical composition ratio of Polygonatum sibiricum roots in Henan compared to other production areas. Both HCA and PCA results can effectively distinguish samples from Hebei, Anhui, and Henan production areas, but the differentiation of samples from Jiangsu production area is not obvious. Phenolic acid components such as chlorogenic acid are easily soluble in water and unstable when exposed to heat. Therefore, processing, storage, and transportation in different production areas can affect the quality of medicinal materials, leading to differences in the quality of standard decoction of Polygonatum sibiricum from different production areas. Using OPLS-DA, three differential biomarkers were identified and ranked according to VIP values, namely peak 9>peak 6>peak 5. This indicates that there are significant differences in peak 9, peak 6, and peak 5 of the standard decoction of Houttuynia cordata from different production areas. It has been confirmed that peak 9 is p-light cinnamic acid, peak 5 is chlorogenic acid, and peak 6 needs further research. At present, many provinces have introduced local policies to encourage fresh cutting of medicinal herbs in their production areas and reduce the loss of effective ingredients. Studies have shown that the content of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid in the slices made by washing and cutting grass coral herbs while they are fresh is significantly higher than that in slices made by traditional processing methods. The anti-inflammatory and analgesic effects of grass coral slices processed while they are fresh are significantly better than those of traditional slices, indicating the feasibility of combining grass coral processing with production area processing. Jiumao root is rich in phenolic acid components, especially suitable for fresh processing in the production area, thereby increasing the content of medicinal materials.

This study established a UPLC fingerprint method for the standard decoction of Polygonatum sibiricum and simultaneously determined four phenolic acid components. The method is easy to operate, stable, reliable, and has good repeatability, providing important reference for the quality evaluation of Polygonatum sibiricum standard decoction, medicinal materials, decoction pieces, and related preparations.