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An approach based on HPLC-fingerprint and chemometrics to quality consistency evaluation of Liuwei Dihuang Pills produced by different manufacturers

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Abstract

Similarity analysis based on fingerprints has been commonly used in quality consistency evaluation of herbal medicines. However, very little efforts have been paid in interpreting of the differences in the fingerprints for the purpose of quality control. In this study, a facile and reproducible HPLC-fingerprint of Liuwei Dihuang Pills (LWPs) was developed. Both similarity analysis and Principal Component Analysis (PCA) were employed to evaluate quality consistencies of LWPs produced by five manufacturers. Contribution plots generated by PCA were performed to interpret differences in the chromatograms of samples from different groups and some peaks which importantly contribute to classification were separated and identified. Then, differences in these samples were verified by quantitative analysis and the way to improve quality consistency was discussed. TCM was usually complex in chemical constituents, it is difficult to identify and quantify all of them. Our study demonstrated that the combination of fingerprints of TCM and PCA not only offered a powerful way to quality consistency evaluation of TCM preparations, but also might simplify the process of quality control by quantitation of some important constituents.

Introduction

The curative effects of Traditional Chinese Medicine (TCM) are principally based on the synergic effect of their multi-targeting, multi-ingredient preparations, in contrast to modern pharmacology and drug development that often focus on a single chemical entity [1]. Therefore, the method employing a few markers or pharmacologically active constituents to assess the quality and authenticity of the complex preparations has a number of severe challenges [2]. Now, chromatographic fingerprint technique plays an important role in controlling the quality of TCM for the systemic characterization of compositions of samples and focusing on the identification and assessment of the stability of the components [3]. Both Food and Drug Administration (FDA) [4] and European Medicines Agency (EMEA) [5] clearly denoted that appropriate fingerprint chromatograms should be applied to assess the consistency of the botanical drugs. Chemometrics is the application of mathematical and statistical techniques to retrieve more information from data. Therefore, State Food and Drug Administration of China (SFDA) suggested that all of herbal chromatograms should be evaluated by their similarities, which derive from the correlative coefficient and/or cosine value of vectorial angle of the original data [6], [7]. Currently, quality consistency evaluation of TCM products or herbal medicines based on similarities among chromatograms is extensively used. But, very little efforts have been paid in interpreting or understanding of the differences in the chromatograms for the purpose of quality control [8].

Liuwei Dihuang Pills (LWPs), one of the classical TCM prescriptions for “nourishing the kidney-yin”, is widely used in preventing and curing many diseases, especially the disorder of immune and endocrine systems such as diabetes, dizziness, tinnitus, etc. [9]. The effectiveness of it has been well documented during long-term clinical practice [10]. LWPs consists of six Chinese herbs including Radix rehmannia, Fructus corni, Rhizoma dioscoreae, Rhizoma alismatis, Cortex moutan radicis and Poria cocos. There are more than twenty pharmaceutical manufactures that produce LWPs in China. According to the Chinese Pharmacopoeia, loganin and paeonal in LWPs are required to be qualified by two different HPLC systems for the purpose of quality control, which is laborious and time-consuming. Some researchers [11], [12], [13] developed HPLC-fingerprints and quantified contents of some bioactive compounds of LWPs. Yet, no reports have been concerned about consistency of it from different suppliers according to their global composition. Therefore, developing a suitable method for quality consistency evaluation of products from different manufactures based on fingerprint is quite necessary.

In the present study, a simple and facile HPLC-fingerprint of LWPs was developed. To assess the quality consistency of LWPs from five manufactures, classification of samples by similarity analysis and Principal Component Analysis (PCA) based on fingerprints was performed. Furthermore, contribution plots generated by PCA were employed to interpret differences in the chromatograms for the classification. With the help of semi-preparative HPLC and HPLC–MS, some peaks which importantly contribute to classification were identified and quantified by standard compounds. The process in this paper was shown in Fig. 1, it is equally applicable to other herbal medicines based on fingerprints in quality assessment for the purpose of quality control.

Section snippets

Reagents and samples preparation

Samples of LWPs from five manufacturers were summarized in Table 1. Loganin was purchased from Sigma Chemical Co. (USA). Paeoniflorin was purchased from Zelang Medical Technology Co., Ltd (Nanjing, PR China). Paeonal was provided by Xi’an Acetan Bio-Tech Co., Ltd. (Xi’an, PR China). Aqueous methanol (50%, v/v) stock solutions containing standard compounds were prepared and diluted to appropriate concentrations for the construction of calibration curves for the quatitative analysis. Acetonitrile

Optimization of the chromatographic system

Optimization of parameters in HPLC was done through investigating the influence of the mobile phase and detection wavelength. In this work, methanol or acetonitrile was chosen as the mobile phase. Considering the complex of the sample, a little amount of phosphoric acid was added to the mobile phase to reduce the ionization and lower the polarity of these compounds. Three concentrations of phosphoric acid (0.06%, 0.03% and 0.015%) with methanol or acetonitrile were investigated. The results

Conclusion

The effective materials exerting efficacy in TCM are complicated, which may be compatible to or may antagonize with each other, and the difference in the effectiveness of the products from different suppliers lies in the variation of the chemical constituents and their relative contents. Thus, simultaneous determination of maximum number of compounds was commonly used strategy for quality control. However, it is difficult to identify and quantify all chemical constituents in TCM. Our study in

Acknowledgement

The authors would like to thank Dr. Yi-Zeng Liang, College of Chemistry and Chemical Engineering, Central South University, Changsha, PR China, for providing the software of Computer Aided Similarity Evaluation System.

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