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Simultaneous determination of eleven characteristic lignans in Schisandra chinensis by high-performance liquid chromatography
Schisandra chinensis, one of the well-known traditional Chinese herbal medicines, is derived from the dry ripe fruits of Schisandra chinensis (Turcz.) Baill. according to the 9th China Pharmacopeia. Lignans are the main components isolated from extracts of S. chinensis and their content varies depending on where S. chinensis was collected. We have established a qualitative and quantitative method based on the bioactive lignans for control of the quality of S. chinensis from different sources.
Materials and Methods:
To develop a high-performance liquid chromatography method, an Elite ODS C18 column (250 mm Χ 4.6 mm, 5μm) at a column temperature of 30°C and flow rate of 1.0ml/min using acetonitrile (A) and water (B) as the mobile phase with a linear gradient and the peaks were monitored at 217 nm.
All calibration curves showed good linearity (r ≥ 0.9995) within test ranges. This method showed good repeatability for the quantification of these eleven components in S. chinensis with intra- and inter-day relative standard deviations less than 0.43% and 1.21%, respectively. In the recovery test, results of accuracy ranged from 99.51% to 101.31% with RSD values less than 2.
The validated method can be successfully applied to quantify the eleven investigated components in 22 samples of S. chinensis from different sources.
Schisandra chinensis; lignans; quality control; high-performance liquid chromatography
Identification and validation of rice reference proteins for western blotting
Journal of Experimental Botany
Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.
Antibody-based proteomics; rice (Oryza sativa L.); reference gene; reference protein; western blotting
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