Based on HPLC fingerprint combined with chemical pattern recognition and multi index component quantitative evaluation of the quality of Houttuynia cordata medicinal materials
The medicinal herb of Peucedamum praeruptorum Dunn is derived from the dried roots of the white flowered Peucedamum praeruptorum Dunn plant in the Umbelliferae family. It was first recorded in the “Record of Famous Doctors” and has the effects of dispersing wind heat, reducing qi, and resolving phlegm. It is commonly used for symptoms such as phlegm heat cough and asthma, yellow thick phlegm, wind heat cough, and excessive phlegm. In terms of modern chemical composition and clinical applications, the active ingredients of Peucedanum praeruptorum are mainly horn type coumarins, including Peucedanum praeruptorum A, Peucedanum praeruptorum B, Peucedanum praeruptorum Ding, etc., which have pharmacological effects such as expectorant, anti heart failure, blood pressure lowering, and anti-inflammatory. Since ancient times, the medicinal herb of Qianhu has mainly been wild resources, distributed in Anhui, Hunan, Jiangxi, Zhejiang, Jiangsu and other places. In recent years, due to the surge in resource demand, artificial cultivation has gradually replaced wild cultivation. Its main cultivation areas are Ningguo in Anhui, Huarong in Hunan, Zunyi in Guizhou, Fengjie in Chongqing, and other places. After the artificial cultivation of white flowered Peucedanum praeruptorum, various production areas have introduced and planted it, ranging from Zhejiang in the east to Yunnan and Sichuan in the west. However, due to the vast differences in production environments, there are significant differences in the quality of Peucedanum praeruptorum medicinal materials, with varying levels of quality. In addition, the artificial planting of white flowered Peucedanum praeruptorum is prone to problems such as early bolting, which forces the early harvesting of Peucedanum praeruptorum medicinal materials. The insufficient growth period leads to a significant increase in the unqualified rate of medicinal materials. The 2020 edition of the Chinese Pharmacopoeia stipulates that “the content of Peucedanum praeruptorum A shall not be less than 0.90%, and the content of Peucedanum praeruptorum B shall not be less than 0.24%”. However, the quality compliance rate of Peucedanum praeruptorum medicinal materials in the market is only 26%, and unqualified Peucedanum praeruptorum medicinal materials flood the market. In addition to cultivating high-quality new varieties, forming suitable planting techniques, and standardizing initial processing in production areas, objective, scientific, and reasonable quality evaluation methods and standards are also important aspects of ensuring the quality of Qianhu medicinal materials. At present, the key to evaluating the quality of Houttuynia cordata medicinal materials lies in the content of berberine and berberine in white flowered Houttuynia cordata. However, this often cannot comprehensively evaluate the intrinsic quality of Houttuynia cordata medicinal materials and directly affects the clinical application of traditional Chinese medicine.

HPLC fingerprint can comprehensively reflect the intrinsic quality of medicinal materials and is a widely recognized quality control method. Combined with multi index content determination, it has become the mainstream of quality control for traditional Chinese medicinal materials. Unsupervised methods such as hierarchical clustering analysis (HCA) and principal component analysis (PCA), as well as supervised chemical modeling methods such as partial least squares (PLS) and artificial neural networks, can better reflect the intrinsic quality of medicinal materials and are widely used new methods in recent years. This study is based on the previous foundation, creating an HPLC fingerprint and combining chemical modeling methods such as cluster analysis, principal component analysis, and partial least squares regression analysis to comprehensively evaluate the medicinal materials of different origins of Houttuynia cordata; At the same time, the content of four coumarin components, namely bergamot lactone, paeoniflorin, paeoniflorin, and paeoniflorin E, was quantitatively determined to clarify the compositional differences of paeoniflorin from different regions, providing reference for the development of quality standards for paeoniflorin.

In order to optimize the chromatographic conditions of the HPLC fingerprint of Houttuynia cordata, this study based on previous research optimized the detection wavelength, mobile phase, and flow rate. The analysis was conducted using full wavelength scanning method, and the results showed absorption at wavelengths of 190-245nm and 290-340nm. Choosing 321nm as the detection wavelength has a large amount of information, good response value, and less interference from impurities. Therefore, 321nm was determined as the detection wavelength. The investigation of the mobile phases “acetonitrile water” and “methanol water” showed that the separation effect of “methanol water” was better, and 0.5% formic acid water could obtain better peak shapes. Therefore, “methanol-0.5% formic acid water” was determined as the mobile phase. In addition, investigations were conducted on flow rates of 1, 0.8, and 0.5mL/min, and it was found that 0.5mL/min had better separation efficiency. Therefore, 0.5mL/min was determined as the flow rate for this method.

This study used HPLC fingerprinting to analyze samples from Hunan, Anhui, Chongqing, Hubei, Guizhou, Yunnan and other production areas. The fingerprints of 16 batches of Chinese medicinal herbs showed high similarity, with a total of 21 common peaks identified and 7 components identified. In chemical pattern analysis, the 16 batches of samples were divided into three categories. Based on reference literature reports, they can be divided into three categories: authentic production areas such as Ningguo and Bozhou in Anhui, Qianhu mainly in Hunan, Guizhou, Chongqing, and high-altitude production areas in Yunnan. Through principal component and PLS-DA analysis, the cumulative variance contribution rate of the six principal components was 86.213%. Further analysis identified eight common peaks that distinguish the origin, which can be used as the main characteristic components of the origin. The content of berberine and bergamot in samples of Houttuynia cordata from high-altitude Yunnan production areas is significantly higher than in other production areas, while berberine is slightly higher than in other production areas. We speculate that high altitude is conducive to the accumulation of bergamot lactone, berberine, and berberine in Houttuynia cordata medicinal materials. Yang et al. believe that high altitude is conducive to the generation and accumulation of Peucedanum praeruptorum and Peucedanum praeruptorum, while Luo et al. believe that altitude has a greater impact on the content of Peucedanum praeruptorum than Peucedanum praeruptorum. Although this study is different from previous research, it can be clarified that high-altitude environments are more conducive to the formation of the quality of Houttuynia cordata medicinal materials.

In the analysis of multi index component content determination, a total of 9 batches of Peucedanum praeruptorum samples met the requirements of the Chinese Pharmacopoeia for Peucedanum praeruptorum A, accounting for 56.25%. However, no batch met the limit requirements for Peucedanum praeruptorum B, with the highest being only 2.01mg/g. Xu et al. and Liu et al. also found that in the medicinal herb of Houttuynia cordata, white flowered Houttuynia cordata A is more likely to meet the standard, while the phenomenon of white flowered Houttuynia cordata B not meeting the standard is more common. White flowered Houttuynia cordata is a perennial plant that blooms once. Large scale planting has led to a change to an annual variety, and insufficient age is an important reason for the decline in the quality of Houttuynia cordata medicinal materials. In addition, white flowered Houttuynia cordata is prone to early bolting, and “gunpowder seeds” (annual seeds) flood the market. Through long-term iterative selection, the quality of Houttuynia cordata medicinal materials has been artificially selectively declining year by year. Further research is needed to determine whether the widespread decline in the quality of Qianhu medicinal herbs affects clinical efficacy.

According to literature reports, there is a certain degree of negative correlation between the content of Peucedanum praeruptorum A and Peucedanum praeruptorum B. This study also found this trend, but the specific reasons need to be further confirmed. The reasons for the poor quality of Chinese medicinal herbs in the market, in addition to ecological environment, genetic factors, and artificial intervention, may also be closely related to the current quality evaluation. In the current standards, the qualification rate of pre sales Hu medicinal materials in the market is extremely low, especially the compliance rate of the content of Baihua Qianhu Yi, which is no longer in line with the clinical use and industrial development of Qianhu medicinal materials. In this context, Qiu et al. explored the use of the ratio or sum of Peucedanum praeruptorum A and Peucedanum praeruptorum B to comprehensively evaluate the quality of Peucedanum praeruptorum medicinal materials. Liu et al., Xiao et al., Shi et al. all conducted evaluation studies on the fingerprint spectrum of Peucedanum praeruptorum medicinal materials from different perspectives and achieved good expectations.

This study used a combination of fingerprint analysis, multi index component analysis, and chemical model analysis to analyze and evaluate the quality of medicinal materials, which has important reference significance for the formulation of quality evaluation standards for Qianhu medicinal materials. However, the shortcoming is that the samples collected in this study were mainly obtained from markets, planting bases, and other sources, without detailed records of germplasm, ecological environment, planting techniques, and other information. The limitation of sample consistency may have some impact on this study. In subsequent research, efforts should be made to strengthen the study of the impact of ecological environment on the quality of Houttuynia cordata, address the core issue of early bolting of white flowered Houttuynia cordata, increase the sample size of quality standard research, and develop quality evaluation standards that meet market expectations and promote sustainable development of the industry.