Simultaneous quantification of 12 bioactive components of Ligusticum chuanxiong Hort. by high-performance liquid chromatography
Introduction
Rhizoma Chuanxiong, derived from the rhizome of Ligusticum chuanxiong Hort. (Umbelliferae), is a well-known traditional Chinese medicinal (TCM) herb with haemodynamic and analgesic effects [1]. In the TCM practice, this herb is commonly prescribed for the treatment of migraine and various cardiovascular diseases, such as angina pectoris and ischemic stroke [2], [3].
From Rhizoma Chuanxiong, more than 30 compounds, which belong to various different structural types, have been isolated, and among them, several components belonging to three types, namely phenolic acid, alkaloid, and phthalide, have been found to be pharmacologically active. For instance, the phenolic acid-type compound: ferulic acid and coniferylferulate; the alkaloid-type compound: tetramethylpyrazine (TMP); and the phthalide-type compound: Z-ligustilide, senkyunolide A, 3-butylidenephthalide and levistolide A, have been reported to be the biologically active components contributing to the therapeutic effects of this medicinal herb [4], [5], [6], [7], [8].
As a naturally occurring medicinal herb, the type and quantity of the bioactive ingredients in Rhizoma Chuanxiong vary with the growth environments and post-harvesting process [9]. Furthermore, coniferylferulate and several phthalides are thermolabile [10], [11], [12], [13], which may also influence the quality of this herb and consequently its therapeutic outcomes if the post-harvesting process alters. Therefore, a reliable quality control method is needed for the qualitative and quantitative determination of the main bioactive components of Rhizoma Chuanxiong.
To date, various analytical methods have been reported for the analysis of the active ingredients of Rhizoma Chuanxiong, including GC–MS [14], [15], HPCE-UV [16], HPLC–MS [15], [17], and HPLC-UV [15], [17], [18], [19]. However, the elevated temperatures used in all reported GC methods may not be appropriate for quantification of the thermolabile compounds such as Z-ligustilide, coniferylferulate, senkyunolide A and 3-butylidenephthalide [10], [11], [12], [13]. On the other hand, all the published HPCE and HPLC methods were unable to simultaneously determine all three types of the active ingredients present in Rhizoma Chuanxiong. Therefore, the present work aimed to develop an analytical method to simultaneously determine all reported main active compounds. A simple HPLC-UV assay using an internal standard method has been developed, for the first time, to simultaneously determine and quantify twelve ingredients, which belong to the three active types and are the reported bioactive components present in Rhizoma Chuanxiong. The chemical structures of these 12 compounds are illustrated in Fig. 1. The developed method has been subsequently applied to analyze six different Rhizoma Chuanxiong samples for the quantification of these twelve components.
Section snippets
Chemicals and standards
HPLC grade methanol (E. Merck, Darmstadt, Germany) was used for the HPLC analysis. Analytical-reagent grade ethanol was used for the extraction of Rhizoma Chuanxiong. Vanillin from Fluka Chemie (Buchs, Switzerland), ferulic acid and TMP from Acros Organics (Geel, Belgium), Z-ligustilide from ChromaDex (Santa Ana, CA, USA), sedanolide and α-naphthaflavone (utilized as internal standard) from Sigma (St. Louis, MO, USA), and 3-butylidenephthalide from Aldrich Chemical Company (St. Louis, MO, USA)
Results and discussion
As shown in Fig. 2, all 12 analytes were eluted with baseline separation under the optimized chromatographic condition. Since the 12 ingredients have their maximum UV absorptions (λmax) at different wavelengths, in order to obtain the highest sensitivity to analyze each analyte, four different wavelengths at 254, 274, 284 and 294 nm, respectively, were chosen to detect and quantify all 12 analytes simultaneously. The wavelengths for monitoring different analytes are listed in Table 1. A further
Conclusions
This is the first report on the development of a simple, sensitive and specific HPLC-UV method to simultaneously quantify all main bioactive ingredients in Rhizoma Chuanxiong. The results demonstrate that the developed method was accurate and reproducible. This method has also been applied successfully to simultaneously quantify twelve constituents in six Rhizoma Chuanxiong samples. The results demonstrate that this method could be readily utilized as a suitable quality control method for
Acknowledgements
The authors gratefully acknowledge the financial support of ITF fund (UIM/34) from Innovative Technology Council of The Hong Kong SAR Government and Kinetana Hong Kong Herbal Pharmaceuticals Limited.
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