Prediction and analysis of quality biomarkers for Lonicera japonica based on high-performance liquid chromatography fingerprint and network pharmacology
Honeysuckle is sweet, cold, and belongs to the lung, heart, and stomach meridians. It has the effect of clearing heat and detoxification, and evacuating wind heat. It is used for carbuncle, swelling, sore throat, erysipelas, hot blood dysentery, wind heat, cold, and febrile diseases. At present, there are more than 80 prescriptions of traditional Chinese patent medicines and simple preparations of honeysuckle in the Chinese Pharmacopoeia, such as Ganmao Anticough Tablets, Kouyan Qingpian, Ganmao Anticough Capsules, Qinggan Lidan Granules, Yinxieling Granules, and so on. Shanyinhua is not only a commonly used raw material for bulk Chinese medicinal herbs and formulations, but also a raw material for chemical spice extraction. At the same time, it is also developed as a tea and skincare product, with broad market application prospects.
Modern research has shown that the chemical components of Lonicera japonica are numerous and complex, including organic acids, triterpenoid saponins, flavonoids, volatile oils, and trace elements. At present, the Chinese Pharmacopoeia only uses chlorogenic acid, Lonicera japonica saponin B, and Panax notoginseng saponin B as quality evaluation indicators for Lonicera japonica, and only sets limits for three chemical components, including organic acids and saponins. A large number of studies have shown that flavonoids such as verbascoside are also the material basis for the medicinal effects of Lonicera japonica. The chemical composition of traditional Chinese medicine is complex, and the medicinal effects do not only rely on a single chemical component, but also on the common additive effects between multiple components. Therefore, only a few components are used to evaluate the quality of medicinal materials, which is not comprehensive enough.
In order to improve the quality standards of traditional Chinese medicine and products, Academician Liu Changxiao proposed the concept of traditional Chinese medicine quality marker (Q-Marker) in 2016 based on the analysis of existing quality evaluation and control methods. The quality indicators of traditional Chinese medicine have five principles: transmission and traceability, uniqueness, effectiveness, measurability, and compound compatibility. They can be used as comprehensive quality evaluation and control indicators for traditional Chinese medicine and products. After the concept of Q-Marker was proposed, it has been widely used to analyze the intrinsic chemical quality attributes of traditional Chinese medicine that are closely related to the effectiveness of Chinese herbal medicines, decoction pieces, and formulations, providing new ideas for the whole process quality control and quality traceability of Chinese herbal medicines and their products.
Longhui and Xupu are the main production areas of Hunan gray felt honeysuckle, which is divided into two categories based on whether the corolla is unfolded in the later stage: multicolored flower (corolla unfolded) and Xianglei (corolla not unfolded). Currently, the primary processing methods in the production area mainly include steam withering and low-temperature drying, resulting in four specifications of Xiangxian mountain silver flower medicinal materials in the market: steam withering Xianglei, low-temperature drying Xianglei, steam withering multicolored flower, and low-temperature drying multicolored flower. To evaluate the quality of different specifications of Hunan produced Shanyinhua and establish a comprehensive quality evaluation index, this article is based on the concept of traditional Chinese medicine quality markers, combined with research methods such as HPLC fingerprint, multivariate statistical analysis, and network pharmacology to predict and verify the Shanyinhua Q-Marker, in order to provide ideas and references for the quality evaluation and quality control of Shanyinhua.

 

Discussion on quality markers based on the measurability of chemical composition. This article established a fingerprint of Lonicera japonica by HPLC, and the similarity between 29 batches of Lonicera japonica samples was greater than 0.95. The methodological investigation results proved that the HPLC fingerprint was stable and reliable. The content determination of potential quality markers for Lonicera japonica was carried out, and the results showed that all 8 components above could be detected in all samples. Moreover, the content of chlorogenic acid, Lonicera japonica saponin B, and Panax notoginseng saponin B in all samples was much higher than the pharmacopoeia standard, indicating that the quality of different specifications of Lonicera japonica produced in Hunan is good. The HCA analysis results showed that at a classification distance of 20, except for sample 18, the other samples were clustered into two categories according to processing methods (steam killing, low-temperature drying); At a classification distance of 6, samples other than 1, 27, 13, and 18 were grouped into four categories based on their varieties and processing methods (steam killed Xianglei, steam killed Wucai Hua, low-temperature dried Xianglei, low-temperature dried Wucai Hua). Among them, 1 and 27 are steam killed Xianglei, but they are closer in classification to low-temperature dried Xianglei in HCA analysis. 18 is steam killed Wucai Hua, which is closer in classification to low-temperature dried Wucai Hua. 13 is steam killed Xianglei, which is closer in classification to steam killed Wucai Hua, indicating that processing methods have a significant impact on the quality of medicinal materials.

Discussion on quality markers based on their effectiveness. When Shanyinhua is used as medicine, it has a sweet and cold taste, and is beneficial for the lungs, heart, and stomach meridians. It has the effects of clearing heat, detoxifying, and dispersing wind and heat. Modern research has shown that the sweet taste is related to components such as saponins, vitamins, proteins, sterols, and amino acids. It is also related to clearing heat and detoxifying, dispersing wind heat, and enhancing immunity through anti-inflammatory, antibacterial, and immune responses in modern research. Using network pharmacology analysis from an effectiveness perspective, it was found that eight compounds, including caffeic acid and swertiamarin, regulate 16 key targets including STAT3, EGFR, MAPK1, APP, PIK3CA, ITGB1, CASP3, and act on physiological processes such as energy metabolism, neurotransmitter transmission, and inflammatory response. These compounds are related to their traditional functions of clearing heat and detoxifying, and dispersing wind heat. Through in-depth analysis of relevant targets, it can be found that there are particularly many targets for inflammatory response, taking STAT3 and EGFR targets as examples. The full name of STAT3 target is Signal Transformer and activator of transcription 3, which refers to signal converters and transcriptional activators. Members of the STAT family act as cytokines and growth factors, and this protein can be phosphorylated and activated by various cytokines and growth factors, including EGF, IL5, IL6, HGF, LIF, and BMP2. The full name of EGFR target is epidermal growth factor receptor, which is a receptor for members of the epidermal growth factor family. EGFR induces receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation. EGFR is a component of a severe form of cytokine storm that causes COVID-19 infection caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Research has confirmed that the above targets are indeed prominent targets in lung related infections, consistent with the traditional taste and meridian tropism of mountain silver flowers. The literature shows that caffeic acid can inhibit the production of TNF – α and PGE2 through NF kB dependent pathway, increase the bacterial clearance activity of macrophages, inhibit the formation of AGE, regulate intestinal inflammation, reduce the activities of ACE and arginase in serum and kidney, reduce MDA and increase the level of NOx to fight hypertension, block STAT3 and inhibit MMP2 and MMP9, prevent the production of ROS and reduce the angiogenesis of tumor cells to play an anti hepatoma role, activate JNK/Bcl-2 mediated autophagy in vitro to reduce A53T α – synuclein, improve the behavior of Parkinson’s disease, protect dopaminergic neurons, reduce oxidative stress to improve blood lipid levels, and reduce aortic injury to fight atherosclerosis. Zhangcai glycoside can exert anti fibrotic effects by upregulating miR-29a, inhibiting col1 and TIMP1, inhibiting the activation of NLRP3 inflammasome to prevent non-alcoholic fatty liver disease, inducing Akt phosphorylation in liver cancer cells, and inhibiting Pck1 expression to combat cancer. Through metabolomics analysis, isochlorogenic acid A, isochlorogenic acid B, chlorogenic acid, neochlorogenic acid, and Chuanshenduan saponin B are all blood components of Lonicera japonica. Therefore, literature research results and network pharmacology studies have shown that the above 8 potential quality markers are indeed the material basis for the pharmacological effects of Lonicera japonica.
In summary, this study, based on HPLC fingerprint and cluster analysis, can effectively distinguish different varieties and processing methods of mountain silver flowers; Based on the OPLS-DA analysis results and combined with network pharmacology, this study elucidated that eight compounds including chlorogenic acid, isochlorogenic acid A, neochlorogenic acid, swertiamarin, isochlorogenic acid B, Lonicera japonica saponin B, caffeic acid, and Panax notoginseng saponin B can be used as quality markers for Lonicera japonica, providing reference for further establishing and improving the quality standards of Lonicera japonica.