Research on UHPLC Fingerprint and Chemical Pattern Recognition of Gouteng
The 2020 edition of the Chinese Pharmacopoeia includes Uncaria rhynchhophylla (Miq.) Miq.ex Havil Uncaria macrophylla Wall, Uncaria hirsuta Havil Dried hooked stems and branches of Uncaria sinensis (Oliv.) Havil. or Uncaria sessilifructus Roxb. At present, the main medicinal vines circulating in the pharmaceutical market are Uncaria rhynchhophylla (Miq.) Miq.ex Havil. Gouteng has the effects of clearing heat and calming the liver, calming the wind and calming the startle. It can be used for liver wind internal movement, epileptic seizures, febrile seizures, cold shock, pediatric crying, pregnancy eclampsia, headache and dizziness, etc. It is commonly used in clinical practice to treat diseases such as hypertension, arrhythmia, anxiety, epilepsy, etc. Gouteng contains numerous chemical components, mainly including alkaloids, flavonoids, triterpenoids, etc. Among them, alkaloids are considered the main pharmacological components of Gouteng for lowering blood pressure.

The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable chromatogram that provides chemical composition characteristics. As for the medicinal herb Gouteng, many scholars have conducted research on the fingerprint spectrum of Gouteng. For example, Huang et al. established HPLC chromatographic fingerprint spectra of Uncaria from different regions using Uncaria alkaloids and Isocrocea alkaloids as indicator components, while Tan et al. established HPLC fingerprint spectra of Uncaria alkaloids from different regions. These studies have made useful explorations for the in-depth evaluation of the quality of Gouteng, but due to the focus on a few alkaloid components such as Gouteng alkaloids and IsoGouteng alkaloids, and only using a single similarity evaluation, there is still room for improvement in accurately and comprehensively reflecting the internal quality of Gouteng without deeper mining and exploration of fingerprint data.

In order to objectively, comprehensively, and efficiently evaluate the quality of Gouteng medicinal materials, this experiment established fingerprint spectra of 30 batches of Gouteng from different sources using UHPLC. Fifteen common peaks were calibrated using the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software”. After comparison with reference standards, seven common peaks were identified, including chlorogenic acid, isocaprylic acid, isocaprylic acid, Gouteng alkaloid, sewing seed methyl ether, dehydrogenated Gouteng alkaloid, and Gouteng alkaloid. Based on the similarity evaluation of fingerprint spectra, the fingerprint data is combined with chemometric methods, and multivariate statistical methods such as cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) based on chemometrics are applied to analyze and evaluate the quality of Gouteng medicinal materials, in order to screen and identify characteristic compounds of Gouteng quality differences and provide reference for effective quality control of Gouteng.

This study investigated the effects of extraction time (ultrasound 20, 40, 60 min), extraction solvents (100%, 75%, 50% methanol and 100%, 75%, 50% ethanol), chromatographic columns (ACQUITY UPLC HSS T3, Cortecs UPLC T3, Cortecs UPLC C18, and ACQUITY UPLC BEH C18), and different mobile phase systems (methanol-0.1% formic acid aqueous solution and acetonitrile-0.1% formic acid aqueous solution) on the number and separation of chromatographic peaks. Finally, 75% methanol ultrasound extraction for 40 min was determined as the preparation method for the test sample, and ACQUITY UPLC HSS T3 was selected as the chromatographic column, eluted with acetonitrile-0.1% formic acid aqueous solution in a gradient manner. At the same time, different detection wavelengths such as 254, 274, and 366 nm were examined, and it was found that there were many peaks in the chromatogram at 254 nm with good separation.

The quality control of Gouteng medicinal materials in the 2020 edition of the Chinese Pharmacopoeia is qualitatively identified using TLC method with isorhynchophylline as the indicator component. This method is cumbersome to operate and has low sensitivity, making it difficult to comprehensively control the quality of hooked vines. This study first established fingerprint spectra of 30 batches of hook vine from different commercial sources for similarity evaluation, and then conducted chemical pattern recognition research on them. According to the similarity evaluation software, the results showed that the similarity between 30 batches of Gouteng medicinal materials ranged from 0.850 to 0.995; A total of 15 common peaks were identified in the fingerprint spectrum, and 7 chemical components were identified through comparison with reference standards, namely chlorogenic acid, isophenanthroline, isorhynchophylline, rhynchophylline, selegiline methyl ether, dehydrorhynchophylline, and rhynchophylline. Chlorogenic acid has a wide range of pharmacological effects such as antibacterial, antiviral, anti-tumor, lipid-lowering, and blood pressure lowering; Isocamptothecin has anti proliferative effects on human lymphocytic leukemia T cells; Isorhynchophylline and rhynchophylline are the main active ingredients of rhynchophylline in the treatment of hypertension; Cesare methyl ether is an effective 5-HT1A receptor agonist and the main component of traditional Japanese medicine Yigan San; Dehydrohydroquercetin is an effective component of neuroprotective effect on nerve cells; Uncaria alkaloid has anticancer effect, which can inhibit the viability of human breast cancer MCF-7 cells in vitro, and induce DNA damage response and oxidative stress response. Unsupervised cluster analysis and principal component analysis were used to further divide the 30 batches of samples into different groups, and differential evaluation was conducted while evaluating the consistency of the quality of Gouteng medicinal materials; And use orthogonal partial least squares discriminant analysis to screen chromatographic peaks that have a significant impact on sample grouping. Through the combination of fingerprint similarity evaluation and pattern recognition methods, we found that the main factors causing batch differences were 1, 3 (chlorogenic acid), 7 (isocaprylic acid), 9, and 12 (selegiline methyl ether). This suggests that these differential components should be given special attention when evaluating the quality of Gouteng medicinal materials. It is necessary to combine mass spectrometry, nuclear magnetic resonance and other techniques or use relevant reference standards to further identify peaks 1 and 9.

The use of chemical pattern recognition technology combined with fingerprint data analysis can provide a more objective and complete evaluation of the authenticity and quality of traditional Chinese medicine. This experiment used a combination of fingerprint similarity evaluation and chemical pattern recognition technology to study different sources of Gouteng. Compared with existing quality evaluation and quality control methods that only rely on content determination or fingerprint analysis, it not only effectively evaluates the consistency of Gouteng medicinal material quality, but also clarifies the main factors that contribute to the quality differences of Gouteng medicinal material. Therefore, it provides richer information for the quality evaluation and quality control of Gouteng, and provides a certain reference for the quality control and subsequent development of Gouteng.