Chemical fingerprinting of Isatis indigotica root by RP-HPLC and hierarchical clustering analysis

Abstract

The aim was to establish a method for extraction and chemical fingerprinting of extracts of Isatis indigotica roots (“Ban–Lan–Gen”) and to apply the method developed to 18 Ban–Lan–Gen samples. RP-HPLC with gradient elution was performed on authentic reference standards of powdered I. indigotica roots, indigotin and indirubin purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) of China. Eighteen “Ban–Lan–Gen” samples (including the reference powdered herb) were bought from Singapore and different regions in China. Comparisons of the chromatograms showed that the samples can be divided into three groups. The chromatograms of the extracts of five samples were found to be similar to that of the extract of the authentic sample. Eight other samples had similar peaks as the authentic sample but the intensities of the peaks were generally lower, except for the peaks between retention times of 10–40 min. Peaks in these regions were more intense than those found in the extract of the authentic sample. Forty-five characteristic peaks could be found in the extracts of all the above samples. Peaks at retention times 52 and 53 min were determined to be indigotin and indirubin, respectively. The remaining four samples had similar chemical fingerprints to each other but were different from that of the authentic sample. Hierarchical clustering analysis gave similar results as the visual comparison. The RP-HPLC method developed allows simple identification and comparisons of I. indigotica roots. This is the first report of hierarchical clustering analysis of I. indigotica root.

Introduction

Isatis indigotica Fort. (Chinese woad) is a biennial herbaceous plant species distributed widely in China and used as a traditional Chinese medicine. It is different from Isatis tinctoria (European woad) which was used for production of blue dye indigo [1]. The root of I. indigotica (Radix Isatidis), named “Ban–Lan–Gen” in China, is used for the treatment of influenza, epidemic hepatitis and epidemic encephalitis B for hundreds of years in China [2]. As one of the most popular herbal medicines, the root of I. indigotica has been recorded in Chinese Pharmacopoeia since 1985 and its purified extracts have been formulated for clinical use [3a].

Many chemical compounds have been isolated from I. indigotica roots, including indigotin, indirubin, isatin, isatan A, isatan B, trytanthrin, purin, isaindigotidione, organic acids and many amino acids [4], [5]. Indigotin and indirubin have been reported to be biologically active. Their structures are shown in Fig. 1. Indirubin has been shown to have an anti-cancer activity in the treatment of chronic granulocytic leukemia [6]. Recently, it was found that indigotin and indirubin were potent aryl hydrocarbon receptor (AhR) agonists [7] and also that the organic acids in I. indigotica roots had in vitro anti-endotoxic action and antiviral action [8]. The alkaloid isaindigotone from I. indigotica was reported [9] to be a scavenger of superoxide generated either by the hypoxanthine/xanthine oxidase system or stimulated human neutrophils. Isaindigotone also inhibited 5-lipoxygenase activity and leukotriene B₄ production in human neutrophils. It is likely that none of the above compounds can fully account for all the activities of Ban–Lan–Gen. Therefore, a holistic approach for quality control is necessary.

It is well known that herbs collected at different times and planted in different regions may differ in the types and quantities of chemical components, thereby affecting the efficacy. Due to its wide applications, I. indigotica roots are in great demand and sometimes in shortage. In the official Chinese Pharmacopoeia, Baphicacanthus cusia is listed as “Nan–Ban–Lan–Gen” [3b] and often sold as a “Ban–Lan–Gen” substitute. Scientific methods are needed to control the quality of I. indigotica roots. One common method used by the industry is to quantify indigotin and indirubin [10]. Although it has been the adopted method for quality control, the presence of the chemical markers does not always guarantee identity and efficacy, especially if the chemical markers have been spiked into the product. It is difficult to evaluate a sample without any information of the other components other than one or two marker compounds. Chromatographic fingerprinting is a useful method in the identification and quality control of botanical medicines [11], [12], [13], [14], [15], [16] and HPLC fingerprinting analysis of the roots and leaves of Isatis species has been reported [17], [18]. However, the reported extraction and analysis methods provided incomplete fingerprints of I. indigotica roots. Extraction with water or ethyl acetate gave incomplete components. None of the peaks for I. indigotica were identified in the reported chemical fingerprints.

The objectives of this work were to establish a new and simple HPLC method for the identification and quality evaluation of I. indigotica roots. The chromatograms of extracts of samples from different regions are compared visually and analyzed using hierarchical clustering analysis.