Discovery of discriminatory quality control markers for Chinese herbal medicines and related processed products by combination of chromatographic analysis and chemometrics methods: Radix Scutellariae as a case study

https://doi.org/10.1016/j.jpba.2017.02.004Get rights and content

Highlights

  • A strategy was proposed to discover chemical markers for crude and processed Radix Scutellariae.

  • Contribution of each compound toward discrimination of processed products was calculated.

  • Chemical markers were screened out and quantified by a SSDMC method.

  • PCA, PLS-DA, HCA and SVM were performed to discriminate crude and processed Radix Scutellariae.

Abstract

The processing procedure of traditional Chinese herbal medicines (CHMs) plays an essential role in clinical applications. However, little progress has been made on the quality control of crude and processed products. The present work, taking Radix Scutellariae (RS), wine-processed RS and carbonized RS as a typical case, developed a comprehensive strategy integrating chromatographic analysis and chemometric methods for quality evaluation and discrimination of crude RS and its processed products. Chemical fingerprints were established by high-performance liquid chromatography coupled with photodiode array detector and quadrupole time-of-flight mass spectrometry, and similarity analyses were calculated based on eleven common characteristic peaks. Subsequently, four chemical markers were discovered by back propagation-artificial neural network (BP-ANN) modeling. The selected markers were quantified by the ‘single standard to determine multi-components’ (SSDMC) method, and then the quantitative data were subjected to principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Furthermore, support vector machine (SVM) was employed to predict the different processed products of RS. Finally, a hotmap visualization was conducted for clarifying the distribution of major flavonoids among different drugs. Collectively, the proposed strategy might be well-acceptable for quality control of CHMs and their related processed products from the processing mechanism-based perspective.

Introduction

Chinese herbal medicines (CHMs) are attracting intensive attention due to their long historical applications and reliable therapeutic efficacy. Unlike western herbs, many CHMs are usually subjected to a processing procedure. The herb-processing method, which is based on the dialectical treatments of medical needs and the drugs' characteristics, has been assisting CHMs in exerting reasonable pharmacological effects [1], [2]. There are various traditional ways of processing herbs, such as steaming, stir-frying, baking, and braising, etc. These processes contribute to moderating drastic action, enhancing efficacy, increasing potency and/or relieving toxicity [3].

Although CHMs and their related processed products are derived from the same herbal origin, their pharmacological effects and clinical applications varied greatly from each other [4]. The altered effects have been ascribed to the changed chemical profiles between crude herb and the processing-treated herbs [5], [6]. Therefore, for the purpose of precision quality evaluation of crude herb and processed products, discovery of discriminatory quality control markers is very important for their safe and effective use [7]. However, in most cases, the crude herb and processed products share the same quality specification, mainly due to lack of deep understanding of chemical changes during processing treatment [2], [8].

Radix Scutellariae (RS), derived from the roots of Scutellaria baicalensis Georgi (Family Labiatae), is a famous herbal medicine used in China with the Chinese name of ‘Huangqin’. It has been used for the treatment of various ailments including fevers, diarrhea, ulcers, cancers, and various inflammatory diseases [9], [10], [11], [12]. According to the processing theory of CHMs, RS is traditionally converted into wine-processed RS and carbonized RS. Previous studies had proved that the wine-processed RS was more effective in cleaning the lung fire, the upper-energizer heat and humidity with the aid of wine characteristic [13], [14], and carbonized RS could enhance the effect of hemostatic after carbonization [15]. Phytochemical and pharmacological investigations revealed that the flavonoids such as baicalin, wogonoside, baicalein and wogonin in RS were responsible for the curative effects [16]. Unfortunately, the exact difference of chemical composition in crude and processed RS is not yet clear. In Chinese Pharmacopoeia (2015 Version) [17], the content of baicalin (not less than 8%) is officially set as acceptance criterion for both crude and wine-processed RS, which may not be specific and meaningful.

In this work, a comprehensive strategy integrating chromatographic analysis and chemometrics was firstly proposed to identify quality control markers for crude RS and its related processed products. Firstly, the common peaks from chromatographic fingerprint of 49 batches of crude and processed RS samples were comprehensively characterized by a high-performance liquid chromatography combined with photodiode array detector (HPLC-PAD) and quadrupole time-of-flight mass spectrometry (QTOF-MS) method. Secondly, the principal discriminatory markers of the crude and processed products were discovered by back propagation-artificial neural network (BP-ANN) model. Thirdly, a single standard to determine multi-components (SSDMC) method was developed for quantitative analysis of the selected markers in crude RS, wine-processed RS and carbonized RS. Subsequently, the quantitative data were subjected to principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA), followed by support vector machine (SVM) modelling. Finally, the distribution of identified flavonoids in the three different RS products was analyzed by a hotmap visualization, and three content-specific specifications were accordingly suggested.

Section snippets

Materials and reagents

Twenty-five batches of crude RS were collected from cultivated or commercial sources in China and authenticated by Professor Hui-Jun Li from Department of Pharmacognosy in China Pharmaceutical University. Twelve batches of wine-processed RS were processed according to Chinese Pharmacopoeia (2015 Version). Briefly, the crude RS slices were mixed with Chinese rice wine (100:12.5, drug/wine, w/w), then the moistened slices were stir-fried in a metallic pan at 120–140 °C over a low flame until

Optimization of extraction method and chromatographic conditions

To ensure satisfactory extraction efficiency, variables including extraction method, solvent and extraction time were optimized. 40% methanol, 70% methanol, 100% methanol and ethanol were tested as extraction solvents. Results showed that 70% methanol produced a desirable extraction efficiency for flavonoids. Different extraction methods including sonication, reflux and soak at room temperature were tested. Sonication was chosen as the extraction method for the high efficiency and convenience

Conclusion

The processing procedure confers upon traditional CHMs different but valuable therapic effects. Along with the demand of CHMs for medical care and dietary supplement increases, the quality control of CHMs and their related products has become a critical issue. Based on chromatographic fingerprint analysis and BP-ANN model, baicalein, wogonoside, baicalin and wogonin, the four principal discriminatory compounds were determined as the quality control markers of RS and its related products. The

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81322051), the Natural Science Foundation of Jiangsu Province (No. BK20151442) and the Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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These authors contributed equally to this work.

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