Simultaneous Quantification of Three Chemical Types Bioactive Compounds in Radix Isatidis and Its Relevant Pharmaceutical Dosage Forms by HPLC-DAD

Abstract

A simple and rapid high performance liquid chromatography coupled with diode array detector (HPLC-DAD) method was developed for simultaneous quantification of three chemical types of bioactive compounds including an alkaloid (epigoitrin), six nucleosides (uracil, cytidine, hypoxanthine, uridine, guanosine and adenosine), and four phenylpropanoids (syringin, clemastanin B, indigoticoside A and isolariciresinol) in Radix Isatidis and its relevant pharmaceutical dosage forms. The developed method was successfully applied to analyze 32 batches of samples with good linearity (R 2 , 0.9966-0.9999), precisions (relative standard deviation (RSD), 0.24-4.61%), repeatability (RSD, 0.39-4.93%), stability (RSD, 1.18-4.29%) and recovery (range, 95.4-104.6%) of the eleven compounds. The limits of detection (LOD) and limits of quantification (LOQ) of the method ranged 0.0383-0.1744 and 0.1550-0.5467 μg mL -1 , respectively. The results indicated that the newly established HPLC-DAD method was sensitive, precise, accurate and reproducible. It could be applied to the quality control of Radix Isatidis and its relevant pharmaceutical dosage forms. Keywords: Radix Isatidis, HPLC-DAD, alkaloid, phenylpropanoid, nucleosides IntroductionThe well-known traditional Chinese medicine Radix Isatidis (common name: Banlangen) is the dried root of the plant Isatis indigotica Fort. It belongs to the Cruciferae family and widely distributed in the north and central of China.1,2 Radix Isatidis are one of the most frequently used traditional Chinese medicine for treatment of influenza, bacterial infection, fever and epidemic hepatitis.3 Because of the unique pharmacological activities, Radix Isatidis has been developed into various dosage forms such as granules, tablets, capsules and altapharmas.Chemical studies showed that Radix Isatidis contains various compounds such as alkaloids, phenylpropanoids, nucleosides and organic acids. 4 Epigoitrin as an alkaloid was used as a marker compound of Radix Isatidis in the 2015 edition of the Chinese Pharmacopoeia. Uracil, cytidine, hypoxanthine, uridine, guanosine and adenosine are the main nucleosides in Radix Isatidis. 5The latest studies showed that nucleosides were important bioactive compounds related to anti-inflammatory, immunoregulation, antitumor and antivirus activities. [6][7][8][9] Clemastanin B, indigoticoside A, isolariciresinol and syringin are the major phenylpropanoids isolated from Radix Isatidis. [10][11][12] In our previous studies, 13 we found that clemastanin B and syringin had strong inhibitory effects on influenza FM1. Clemastanin B was found to inhibit different subtypes of human (H1N1, including swineorigin H1N1; H3N2 and influenza B) and avian influenza viruses (H6N2, H7N3, H9N2) at different magnitudes of activity.14 From the latest studies, indigoticoside A could inhibit herpes simplex virus (HSV) and influenza A virus. 15Qualitative and quantitative analysis of major bioactive compounds are very important for the quality control of Radix Isatidis. It is well known that traditi...

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