Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography–electrospray ionization mass spectrometry
Introduction
Silymarin, derived from the milk thistle plant Silybum marianum, has been used widely for centuries for the protection of the liver from toxic substances. It has also been used for the treatment of toxic liver damage and for the therapy of hepatitis and cirrhosis [1], [2], [3], [4], [5]. In addition to its antioxidant properties, it has been reported to have exceptionally high anti-tumor promoting activity [5], [6], [7], [8], [9] and has also been linked to the prevention of skin carcinogenesis [10]. Silymarin primarily consists of an isomeric mixture of active flavonolignans: silychristin (Sc), silydianin (Sd), and two groups of diastereoisomeric flavonolignans, silybin A (Sb A) and silybin B (Sb B), and isosilybin A (ISb A) and isosilybin B (ISb B) [11], [12], [13], [14], [15], [16]. The different isomers of silymarin have been reported to have different biological activities [17], [18], [19], [20], [21], [22], [23], [24]. The chemical structures of the six main active constituents of Silybum marianum are shown in Fig. 1.
Standardized Silybum marianum contains 70–80% silymarin and has been widely adopted for production. The complexity of the silymarin product combined with its unregulated manufacturing process has made it difficult to judge the role of silymarin in the treatment of chronic liver diseases. This has been further compounded by the poor documentation of its ingredients, its source and its extraction process. As a consequence, the lack of regulation in the manufacturing process has resulted in a great deal of variety in the herbs used for extraction. Herb plants harvested in different geological regions and seasons have been well known for affecting the quantities of chemical components and potentially the efficacy of the extracts [23], [25], [26], [27]. The quality control of the starting material and the final standardized extracts needs to be assessed. Since there have been no criteria or guidelines for the expression of the quality of silymarin extracts, it is difficult to interpret the historical clinical efficacy studies, especially those of varied drug products. Furthermore, many pharmacological studies on silymarin conducted using standardized plant extract have failed to identify the manufacturing source of silymarin and to quantitate the silymarin contents, including its individual active components [24], [25], [26], [27], [28], [29], [30], making the evaluation of dose-exposure relationships ambiguous. As a result, the dose-exposure relationships have continued to be poorly defined often representing exposures of mixtures known to have discrete pharmacokinetic properties. Therefore, there is a pressing need for an analytical method that can be used for the quality control of each individual constituent in different silymarin products.
Several chromatographic methods have been reported for the separation or quantitative measurement of individual silymarin. Published methods include those based on thin-layer chromatography (TLC) [31], high-performance liquid chromatography (HPLC) separation with ultraviolet (UV) [15], [32], [33], [34], [35], [36], [37], column-switching with electrochemical [15], mass spectrometry (MS) [36] or tandem mass spectrometry (MS/MS) [16] detections, and capillary electrophoresis (CE) [37]. Recently, Ding et al. reported an HPLC method that separates all six constituents and is detected by a diode-array detector (DAD) [33]. In the proposed method, silybin and isosilybin were used to quantify the concentrations of silybin (A and B) and isosilybin (A and B) in silymarin, respectively. Moreover, the HPLC-DAD assay was considered to be of insufficient sensitivity, especially for the clinical pharmacokinetic study samples; the standard working ranges for the method are: 0.1398–1.398, 0.0846–0.846, 0.1437–1.437 and 0.0885–0.885 mg/mL for the silychristin, silydianin, silybin (A and B) and isosilybin (A and B), respectively.
We have previously reported on a specific and sensitive liquid chromatography/tandem mass spectroscopy (LC/MS/MS) method to characterize all six active components of silymarin in either commercial standardized extract or plasma samples [16]. The purpose of this work was undertaken to develop a sensitive and specific LC/MS method to simultaneously quantify and compare the ratio of six constituents of silymarin in commercial standardized extract. This sensitive method will eventually be employed to study the pharmacokinetics of orally-administered silymarin; to discriminate the active constituents in the drug product as well as in the plasma samples collected post dose.
Section snippets
Reagents and materials
The milk thistle herbal supplements used were standardized extracts from General Nutrition Corp. (GNC) (Pittsburg, PA, USA), Natural Resource Products (Mission Hills, CA, USA), CVS Pharmacy Inc. (Woonsocket, RI, USA), Safeway Inc. (Pleasanton, CA, USA), Spring Valley Herbs & Natural Foods (Springfield, MO, USA) and Rite Aid Corp. (Harrisburg, PA, USA). These extracts were compared to that of the Yiganlin brand from China (Shanghai Wellconie International Pharmaceutical Trading Co., Ltd.,
Results and discussion
Currently, all six individual purified standards are not available for the quantification of silymarin although there is a wealth of literature available. Recently, Ding et al. [33] achieved complete separation for the six constituents with UV detection, but the quantification of diastereomers of silybin and isosilybin was performed using a combination of silybin (A and B) and isosilybin (A and B), respectively. The lack of standards available for the quantification of Sb A, Sb B, ISb A and ISb
Conclusions
A sensitive LC/MS method was developed for the simultaneous determination of six active isomeric flavonolignans in silymarin. The established method has been successfully applied to the identification, quantification and comparison of the active components of silymarin in six commercial products. Silymarin contents varied with respect to different brands of commercial standardized extracts; the ratios of individual constituents were also different. We therefore conclude that silymarin has a
Acknowledgement
This project is funded, in part, under a grant with the Pennsylvania Department of Health. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.
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