Quality Evaluation of Dengzhanhua Standard Decoction Based on Fingerprint Atlas and In Vitro Antioxidant Activity Test
Dengzhan Hua, also known as Dengzhan Xixin, is a plant in the Asteraceae family called Erigeron breviscapus (Vant.) Hand- Mazz.’s dried whole plant is harvested in spring and autumn and included in the 2020 edition of the Chinese Pharmacopoeia. It has the effects of promoting blood circulation, unblocking collaterals, relieving pain, dispelling wind and dispelling cold. Modern pharmacological studies have shown that Scutellaria baicalensis has neuroprotective, cardiovascular and cerebrovascular protective, antioxidant, anticancer, and other pharmacological effects. Cancer, cardiovascular and cerebrovascular diseases are closely related to oxidative damage caused by excessive reactive oxygen species such as superoxide anion radicals, hydroxyl radicals, and lipid radicals in the body. Taking antioxidants appropriately can eliminate excessive free radicals in the body, which is beneficial for human health and disease prevention and control. The literature indicates that Lamplighter has excellent antioxidant activity. The chemical composition of Lamplighter is complex, and the relationship between chemical composition and active dosage effect is not yet very clear. The chemical fingerprint of traditional Chinese medicine has been widely used for quality evaluation of Chinese medicine due to its ability to characterize the substance components contained in Chinese medicine, which conforms to the overall and fuzzy nature of traditional Chinese medicine theory. However, its application is increasingly limited as it only reflects the chemical information of traditional Chinese medicine and fails to establish a relationship with its therapeutic effects. In 2020, the Drug Evaluation Center of the National Medical Products Administration issued the “Technical Guidelines for the Detection of Biological Effects of Traditional Chinese Medicine (Trial)”, advocating the establishment of quality control methods related to activity to ensure the safety and effectiveness of traditional Chinese medicine in clinical applications. The study of the “spectrum effect relationship” of traditional Chinese medicine can closely link the overall characteristic expression of active ingredients with the effects of multiple indicators and targets, and can reasonably determine the pharmacological substance basis, which is also in line with the characteristics of traditional Chinese medicine.

Chinese medicine standard decoction is a single decoction of traditional Chinese medicine prepared by standardized processes, used for standardized clinical medication, ensuring the accuracy of medication and consistency of measurement. It serves as a standard reference for measuring whether Chinese medicine formula particles are basically consistent with clinical decoction. The proposal of standard decoction provides a material benchmark for achieving quality consistency of dosage forms based on traditional Chinese medicine decoction and establishing effective quality evaluation methods. This article takes the standard decoction of Dianzhanhua as the research object, and verifies its antioxidant effect through in vitro experiments of scavenging DPPH and ABTS free radicals. At the same time, its HPLC fingerprint is established and the total flavonoid content is determined; Using bivariate correlation analysis to screen for significantly correlated antioxidant components, cluster analysis (CA) and principal component analysis (PCA) were performed on the screened components, and orthogonal partial least squares discriminant analysis (OPLS-DA) was further used to identify differential components; Evaluate the quality of its antioxidant effect to provide a basis for quality control of Lamplighter and its preparations.

 

 

DPPH and ABTS free radicals are often used as indicators to measure the in vitro antioxidant activity of active ingredients. The DPPH method determines the antioxidant capacity of medicinal materials by binding to substances with lone pair electrons, while the ABTS method determines the antioxidant capacity by binding to hydrophilic and lipophilic substances. The main active ingredients of Scutellaria baicalensis include flavonoids. In this study, the total flavonoid content of 16 batches of Scutellaria baicalensis standard decoction was determined to be 2.85-11.28mg/mL. Its in vitro antioxidant activity was evaluated by scavenging DPPH and ABTS free radicals as antioxidant indicators. The IC50 value of DPPH was 1.00-3.39mg/mL, and the IC50 value of ABTS was 0.254-2.16mg/mL, indicating that the ability of Scutellaria baicalensis standard decoction to scavenge ABTS free radicals is stronger than that of scavenging DPPH free radicals. Using Pearson bivariate correlation analysis, it was found that the two in vitro antioxidant activities (IC50 values) were significantly negatively correlated with the total flavonoid content (P<0.01), with correlation coefficients of -0.714 and -0.841, respectively, indicating that the smaller the IC50 value and the higher the total flavonoid content, the stronger the antioxidant capacity of the extract. Therefore, the content of total flavonoids is one of the indicators that affect the antioxidant strength of Scutellaria baicalensis, and can be used as an indicator to evaluate the antioxidant strength of Scutellaria baicalensis.
In the process of establishing fingerprint spectra, Agilent ZORBAX-SB C18, Agilent ZORBAX Eclipse XDB-C18, and Agilent Extend C18 chromatographic columns were experimentally investigated. Based on the comparison of the number of chromatographic peaks and separation degree, it was found that Agilent ZORBAX Eclipse XDB-C18 had better performance; We investigated different flow rates (0.8, 1, 1.2 mL/min) and found that 0.8 mL/min had a better separation effect; We investigated the binary systems of 0.1% formic acid water methanol, 0.1% formic acid water acetonitrile, and 0.1% formic acid water acetonitrile tetrahydrofuran with different gradients. It was found that the separation effect of 0.1% formic acid water acetonitrile tetrahydrofuran was better and the baseline was relatively stable; Screening the detection wavelength of 200-400nm, the results showed that the chromatographic peaks obtained by changing the wavelength at different time periods were multiple and the baseline was stable. Finally, it was determined that the wavelength was 252nm from 0-9 minutes and 330nm from 9-60 minutes.
In recent years, literature on the fingerprint spectra of medicinal herbs such as Scutellaria baicalensis has mostly used HPLC method, which evaluates the similarity of the fingerprint spectra and determines the common peaks; The literature used CA and PCA for chemical pattern recognition of fingerprint spectra and classified medicinal materials, but did not conduct in-depth analysis of the reasons for differences. On the basis of the fingerprint spectrum of the medicinal herb Lampyridis in the early stage of this article, a fingerprint spectrum of the standard decoction of Lampyridis was established, with a similarity of 0.770-0.993, and 21 common peaks were obtained. Eight known components were identified using the reference substance. This study used bivariate correlation analysis to screen 10 chromatographic peaks, namely peaks 3, 4, and 6 (chlorogenic acid), peaks 7, 11, and 13 (scutellarin B), peak 14 (isochlorogenic acid B), peak 15 (scutellarin A), and peaks 17 and 19 (isochlorogenic acid C), which were significantly negatively correlated with the ability to scavenge DPPH free radicals (i.e., the larger the common peak area, the smaller the IC50 value). The Person correlation coefficients were -0.552, -0.733, -0.626, and -0, respectively 665, -0.537, -0.518, -0.767, -0.754, -0.541, and -0.748 indicate that the corresponding chemical substances are the main active ingredients in scavenging DPPH radicals. Thirteen chromatographic peaks, including peaks 3, 4, 6, 7, 8, 11, 13, 14, 15, 16, 17, 18 (isochlorogenic acid A), and 19, were significantly negatively correlated with ABTS free radical scavenging. The Person correlation coefficients were -0.594, -0.861, -0.761, -0.829, -0.645, -0.583, -0.676, -0.721, -0.740, 0.574, -0.652, -0.720, and -0.518, respectively. The corresponding substances were the main active ingredients for ABTS free radical scavenging. The antioxidant activity (IC50 values) determined by two antioxidant methods were significantly correlated with the known components chlorogenic acid, scutellarin B, isochlorogenic acid B, scutellarin A, and chlorogenic acid C, indicating that these five components are the main active substances in the antioxidant activity of scutellarin.
The experiment used 10 common peaks significantly correlated with DPPH and total flavonoid content as variables, and 13 common peaks significantly correlated with ABTS and total flavonoid content as variables for CA and PCA analysis, respectively. The samples were divided into two categories, namely wild S014 and S016, and the remaining 14 batches of planted samples. It is speculated that it may be related to factors such as the growth environment (light, altitude, latitude and longitude, etc.) and harvesting period of the lantern flowers. At the same time, a scoring model for the standard decoction of Scutellaria baicalensis was established with these components as variables. The ranking of scores for the 16 batches of standard decoction was basically consistent with the ranking of in vitro antioxidant activity. On the basis of CA and PCA, the author further conducted OPLS-DA analysis and identified four main differential components related to the scavenging of DPPH free radicals: peaks 11, 15 (scutellarin), 14 (isochlorogenic acid B), and peak 13 (scutellarin); The four main differential components related to the clearance of ABTS free radicals are peaks 11, 15, 14, and peak 8 (scutellarin). The research results suggest that when controlling the quality of Scutellaria baicalensis, in addition to controlling the high content of Scutellaria baicalensis B (i.e. baicalin), other quality control indicators with strong pharmacological activity should be appropriately added.
In summary, this study combines chemical fingerprinting with biological activity evaluation, and explores the antioxidant active ingredients of the standard decoction of Lampyridis through spectral correlation research. The established method is easy to operate and the results are reliable, which can provide a basis for further research on the pharmacological effects and quality evaluation of Lampyridis. In the future, the sample size can be increased and liquid chromatography-mass spectrometry technology can be used to identify the common peaks of unknown components in the standard decoction, study their pharmacological activities, and comprehensively investigate the spectrum effect relationship of multiple pharmacological activities, providing a basis for the quality research of Scutellaria baicalensis.