Comparative evaluation of different cultivars of Flos Chrysanthemi by an anti-inflammatory-based NF-κB reporter gene assay coupled to UPLC-Q/TOF MS with PCA and ANN

Abstract

Ethnopharmacological relevance

Flos Chrysanthemi (FC), a commonly used traditional Chinese medicine, has five major cultivars (“Boju”, “Chuju”, “Gongju”, “Hangbaiju” and “Huaiju”) from different sources. However, the active constituents of these cultivars have not been studied or characterized with respect to their bioactivity, which is a serious problem when considering quality and safety.

Aim of the study

To evaluate the differences among the five cultivars of FC, and to establish a method for the standardization and quality control of FC related to its bioactivity.

Materials and methods

In this study, the different ingredients in five cultivars of FC were identified by UPLC-Q/TOF and PCA, and the anti-inflammatory ingredients of FC were predicted and screened by artificial neural network (ANN) and an NF-κB luciferase reporter gene assay system. Using this comprehensive method, we successfully screened the anti-inflammatory markers of different cultivars of FC.

Results

Nineteen marker ingredients were confirmed to contribute strongly to the cluster, and eleven compounds in the five cultivars of FC were found to exert potential anti-inflammatory effects. Among these compounds, the NF-κB inhibitor activity of apigenin-7-O-6″-malonyl-glucoside, luteolin-7-O-rutinoside, quercetin-7-O-galactoside, quercetin-3-O-glucoside, apigenin-7-O-rutinoside and apigenin-7-O-glucoside were first reported here. Chlorogenic acid, luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid and luteolin were confirmed to be the most important anti-inflammatory marker ingredients useful for the quality control of FC.

Conclusions

The proposed efficient and systematic method is helpful for the standardization and quality control of FC. Moreover, this comprehensive strategy may prove to be a powerful technique for the rapid establishment of quality control procedures related to bioactivity for other herbal samples and foods.

Introduction

Flos Chrysanthemi (FC) is a medicinal and edible cognate plant widely cultivated in Asia that is botanically known as the dried anthodium of Chrysanthemum morifolium Ramat. In addition to being used for the treatment of a variety of diseases as an essential traditional Chinese medicine (TCM), FC has also been widely used in China as a food supplement, as well as in herbal teas and health foods (Chu et al., 2004, Lai et al., 2007, Lu et al., 2010). Many studies have reported that FC possesses various bioactivities, such as anti-inflammatory (Ukiya et al., 2001), antioxidative (Bor et al., 2006, He et al., 2012), anti-bacterial (Shan et al., 2007), cardiovascular protective (He et al., 2012) and anticancer activities (Xie et al., 2009). According to previous studies, volatile oils, caffeoylquinic acids and flavonoids are regarded as the major constituents of FC and are likely responsible for these aforementioned activities (Kim and Lee, 2005, Lai et al., 2007, Ukiya et al., 2002, Ukiya et al., 2001).

FC has been cultivated for more than 3000 years and is distributed in many regions of China. Due to its different breeds and habitats, FC varies significantly in shapes and chemical compositions. Five major cultivars, “Boju” (BJ, Bozhou, Anhui), “Chuju” (CJ, Chuzhou, Anhui), “Gongju” (GJ, Huangshan, Anhui), “Hangbaiju” (HB, Tongxiang, Zhejiang) and “Huaiju” (HJ, Jiaozuo, Henan), differing with respect to origins and processing method, are used as medicinal herbs. Actually, these five cultivars are not indiscriminated in medicinal and tea usage, and they are all recorded as “Flos Chrysanthemi” in the Chinese Pharmacopoeia (2010 version). Currently, to the best of our knowledge, only chlorogenic acid, luteolin-7-O-glucoside and 3,5-dicaffeoylquinic acid are used as marker compounds to evaluate the quality of FC products by the Chinese Pharmacopoeia. No comprehensive report has yet characterized these cultivars with regard to their bioactivity, which is a serious problem when considering their quality and safety. More seriously, the quality of FC remains variable, and there are a large number of unqualified FC products on the market. Therefore, it is necessary to establish a method for validating the quality of FC related to bioactivity.

In the Chinese Pharmacopoeia, FC is used for “scattering cold”, “cleaning heat and toxin”, and “brightening eyes”. All of these diseases have close relationships with inflammation. Nuclear factor-kappa B (NF-κB) is a transcription factor that is expressed in numerous cell types (Lopez et al., 2014, Panaro et al., 2012). It is involved in the early immune response and inflammatory response, and it lead to the synthesis of cytokines and chemokines such as interleukin (IL)-6, IL-8, RANTES, eotaxin and others (Karin and Greten, 2005). It has been reported that the anti-inflammatory properties of 70% ethanolic extract of FC might result from the inhibition of inflammatory mediators, such as NO, prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), and IL-1β, via suppression of MAPKs and NF-κB-dependent pathways (Cheon et al., 2009). However, a systematic, efficient and rapid method for the screening of NF-κB inhibitors in FC has been obscure until now. Therefore, it is urgent to define the chemical composition and pharmacological characteristics of the five cultivars of FC.

Global analysis techniques, such as chemical fingerprinting by high-performance liquid chromatography (HPLC), often require longer analysis times and involve large amounts of data, even though HPLC has many advantages compared with traditional practice of isolating, purifying, and evaluating the components using various animal experiments (Jiao et al., 2008, Zeng et al., 2012, Zhou et al., 2010). Nowadays, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) is being widely applied to analyze and identify TCM components (Cheng et al., 2013, Ishiuchi et al., 2013, Ma et al., 2013, Qi et al., 2014) owing to its higher peak capacity, greater resolution, increased sensitivity and higher speed of analysis. Moreover, the structural information derived from TOF-MS technology has advantages for accurate mass determination. For further analysis, UPLC–MS data combined with principal component analysis (PCA) for selected marker compounds can be used to classify herbal medicine by specie and origin (Gao et al., 2013, Geng et al., 2013). Over the last decades, the artificial neural network (ANN) has shown its potential as a tool for evaluating the origin, authenticity, species and quality of TCM due to its objectivity and closer to the brain thinking, which make the results accurate and reliable (Lim et al., 2011, Tang et al., 2012, Tang et al., 2004). On the other hand, high-throughput screening based on biological systems (e.g., DNA, proteins, and cells) is also a rapid process of assaying a large number of potential effectors of biological activity against targets (Cheng et al., 2012, Halai and Cooper, 2012, Warzecha et al., 2012). The strategy of combining UPLC-Q/TOF with high-throughput screening has the advantage of describing both chemical and bioactivity characteristics simultaneously, and it has been proven to have high specificity and low sample consumption.

In this study, we developed a tandem method that integrates UPLC-Q/TOF with PCA to classify fifty batches of FC and to identify the different compositions of BJ, CJ, GJ, HB and HJ. Moreover, an artificial neural network (ANN) and an NF-κB luciferase reporter gene assay system were used to predict and verify the potential anti-inflammatory markers of FC. In brief, we established a comprehensive and efficient method suitable for screening compounds that could be used for the quality control of FC as well as other herbs and foods.